Antitumor agent and formulation

Abstract

[Problem] To provide a novel antitumor agent and a compounding agent. [Solution] Provided is an antitumor agent that contains as an active ingredient a glutathione concentration-decreasing agent or a glutathione S-transferase inhibitor and that is administrated at the same time as an effective amount of a compound (II), an antitumor agent that contains as an active ingredient a compound (II) and that is administered at the same time as an effective amount of a glutathione concentration-decreasing agent or a glutathione S-transferase inhibitor, or an antitumor agent that contains as active ingredients a compound (II) and a glutathione concentration-decreasing agent or a glutathione S-transferase inhibitor. (In the formula, R5 is a C1-6 straight-chain or branched alkyl group; R6 is hydrogen or halogen; R7 is a C1-6 straight-chain or branched alkyl group optionally bearing a substituent, the substituent being hydroxy or phenyl; and R8 is hydrogen or halogen.)

Description

Technical Field

[0001] The present invention relates to an antitumor agent and a formulation.

Background Art

[0002] In cancer treatment, the existence of cells resistant to treatments such as anticancer agents and radiation causes recurrence and metastasis, hindering cancer treatment. In recent years, the existence of cancer stem cells has attracted attention as such treatment-resistant cells. Cancer stem cells are highly resistant to various stresses, and the development of drugs targeting cancer stem cells is an urgent task for the radical cure of cancer. However, the analysis of the molecular mechanism of stress resistance in cancer stem cells for the development of cancer stem cell-targeted treatments has just begun.

[0003] CD44, which is one of the markers of epithelial cancer stem cells, is known as a molecule involved in its stress resistance (Cancer Cell. 2011 Mar 8;19(3):387-400). CD44 has splice variant forms (hereinafter, CD44v), and CD44v stably expresses the cystine transporter xCT on the cell membrane. xCT has a function of taking up cystine into the cell, and the cystine taken up thereby is used for the production of glutathione (GSH). Therefore, in cells highly expressing CD44v, the amount of GSH increases. Since GSH has a strong antioxidant action and plays a role in reducing stress generated in cells, cancer stem cells highly expressing CD44v are considered to be resistant to treatment.

[0004] On one hand, there is Sulfasalazine (also known as Salazosulfapyridine, Salazopyrin, and Salicylazosulfapyridine), which is a drug used in the treatment of ulcerative colitis and rheumatoid arthritis. Sulfasalazine is an acidic azo compound of sulfapyridine and 5-aminosalicylic acid (5-ASA). When administered orally, it is decomposed by intestinal bacteria in the intestine into sulfapyridine and 5-aminosalicylic acid (5-ASA). For the above-mentioned diseases, 5-ASA is regarded as the main active ingredient in particular.

[0005] In recent years, it has been clarified that Sulfasalazine in its unchanged form before decomposition has an xCT inhibitory effect and is effective as an antitumor agent (Leukemia vol.15, pp.1633-1640, 2001). That is, when Sulfasalazine is added to cancer cells, the uptake of cystine into cells by xCT is suppressed, the amount of glutathione production decreases. As a result, the oxidative stress resistance of cancer cells decreases, and the sensitivity to antitumor agents increases.

[0006] It is known that Sulfasalazine, which has an xCT inhibitory effect, effectively suppresses the proliferation of cancer stem cells that highly express CD44v (Japanese Patent Laid-Open No. 2012-144498).

Summary of the Invention

Problems to be Solved by the Invention

[0007] An object of the present invention is to provide a novel antitumor agent and a formulation.

Means for Solving the Problems

[0008] The inventors of the present invention have found that sulfasalazine has an antitumor effect alone against tumors composed mainly of undifferentiated tumor cells, but although it reduces cancer stem cells that highly express CD44v in differentiated tumors including tumor cells showing a differentiated phenotype, it has no effect of reducing the volume of the whole tumor. Therefore, in order to obtain an antitumor agent for differentiated tumors by developing an agent having an antitumor effect against tumor cells against which sulfasalazine has no antitumor effect for such differentiated tumors, the inventors made intensive efforts. As a result, it was found that when an aldehyde dehydrogenase inhibitor or oxyphedrine is used in combination with sulfasalazine or L-buthionine-sulfoximine, they have a remarkable antitumor effect against tumor cells that are weakly effective when sulfasalazine or L-buthionine-sulfoximine is used alone, leading to the completion of the present invention.

[0009] One embodiment of the present invention is an antitumor agent containing, as an active ingredient, a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, which is administered simultaneously with an effective amount of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmaceutically acceptable salt thereof.

[0010] JPEG0007683931000001.jpg44170(In the formula, R 5 is a linear or branched alkyl group having 1 to 6 carbon atoms, R 6 is hydrogen or a halogen, R 7 is a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, and the substituent is hydroxy or phenyl, and R 8 is hydrogen or a halogen.) Another embodiment of the present invention is an antitumor agent containing, as an active ingredient, an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmaceutically acceptable salt thereof, which is administered simultaneously with an effective amount of a glutathione concentration reducing agent or a glutathione S-transferase inhibitor.

[0011] JPEG0007683931000002.jpg44170(In the formula, R 5 is a linear or branched alkyl group having 1 to 6 carbon atoms, R2 and R 3 is independently selected from a C1-6 straight-chain or branched alkyl group, or R 2 and R 3 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group having N to which they are attached as a heteroatom, and R 4 is hydrogen or halogen.) In any of the above anti-tumor agents, the glutathione concentration reducing agent may be a drug that inhibits the activity of any of xCT, Thioredoxin-1 (thioredoxin-1: TRX-1), glutamate-cysteine ligase (GCL) (also called γ-glutamylcysteine synthetase) (EC 6.3.2.2), and glutathione synthetase (EC 6.3.2.3). The drug may be an inhibitor of xCT or GCL, and may be sulfasalazine or L-buthionine-sulfoximine. The compound represented by the formula (II) may be oxyfedrine.

[0012] A further embodiment of the present invention is a formulation containing an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor as active ingredients.

[0013] JPEG0007683931000003.jpg44170(wherein R 5 is a C1-6 straight-chain or branched alkyl group, R 6 is hydrogen or halogen, R 7 is a C1-6 straight-chain or branched alkyl group which may have a substituent, the substituent being hydroxy or phenyl, and R 8 is hydrogen or halogen.) In the above-mentioned compounding agent, the glutathione concentration reducing agent may be a drug that inhibits the activity of any one of xCT, Thioredoxin-1 (thioredoxin-1: TRX-1), glutamate-cysteine ligase (GCL) (EC6.3.2.2) (also called γ-glutamylcysteine synthetase), and glutathione synthetase (EC6.3.2.3). The drug may be an inhibitor of xCT or GCL, and may be sulfasalazine or its derivative, or L-buthionine-sulfoximine. The compound represented by the formula (II) may be oxyfedrine.

[0014] A further embodiment of the present invention is an antitumor agent containing any of the above compounding agents.

[0015] Any of the above antitumor agents may be an antitumor agent against tumors containing tumor cells resistant to a glutathione concentration reducing agent or a glutathione S-transferase inhibitor. Aldehyde dehydrogenase may be highly expressed in the tumor cells. The tumor may further contain tumor cells expressing CD44v.

[0016] A further embodiment of the present invention is a measurement method including the steps of simultaneously administering an aldehyde dehydrogenase inhibitor or compound (III) or a pharmaceutically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor to tumor cells in vitro, and measuring the growth rate or cell viability of the tumor cells.

[0017] JPEG0007683931000004.jpg42170(wherein X is hydrogen, halogen, -NH 2 , or -CN, Y is a linear or branched alkyl group having 1 to 6 carbon atoms, and Z 1 and Z 2 are each hydrogen or halogen, and a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, the substituent being hydroxy or phenyl, or Z 1 and Z 2combine together to form an azacycloalkyl group of a 4-membered ring, 5-membered ring, 6-membered ring, or 7-membered ring having N as a heteroatom to which they are attached.) In this measurement method, the tumor cells may be resistant to a glutathione concentration reducing agent or a glutathione S-transferase inhibitor.

[0018] A further embodiment of the present invention is a method for identifying a drug having a combined effect with a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, the method comprising simultaneously administering in vitro to tumor cells a specific glutathione concentration reducing agent or glutathione S-transferase inhibitor and each of a plurality of aldehyde dehydrogenase inhibitors or compound (III) or a pharmacologically acceptable salt thereof, and measuring the growth rate or cell viability of the tumor cells.

[0019] JPEG0007683931000005.jpg42170(wherein X is hydrogen, halogen, -NH 2 , or -CN, Y is a C1-6 straight-chain or branched alkyl group, and Z 1 and Z 2 are each hydrogen or halogen and a C1-6 straight-chain or branched alkyl group which may have a substituent, the substituent being hydroxy or phenyl, or Z 1 and Z 2 combine together to form an azacycloalkyl group of a 4-membered ring, 5-membered ring, 6-membered ring, or 7-membered ring having N as a heteroatom to which they are attached.) A further embodiment of the present invention is a method for identifying a glutathione concentration reducing agent or a glutathione-S-transferase inhibitor having a combined effect with an antitumor agent, compound (III) or a pharmacologically acceptable salt thereof, the method comprising simultaneously administering in vitro to tumor cells the compound (III) and a plurality of glutathione concentration reducing agents or glutathione S-transferase inhibitors, and measuring the growth rate or cell viability of the tumor cells.

[0020] JPEG0007683931000006.jpg42170(wherein X is hydrogen, halogen, -NH 2 , or -CN, Y is a straight-chain or branched alkyl group having 1 to 6 carbon atoms, and Z 1 and Z 2 are each hydrogen or halogen, and a straight-chain or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, where the substituent is hydroxy or phenyl, or Z 1 and Z 2 together form a 4-membered, 5-membered, 6-membered, or 7-membered azacycloalkyl group having N to which they are attached as a heteroatom.) The compound (III) may be the compound (II).

[0021] JPEG0007683931000007.jpg44170(wherein R 5 is a straight-chain or branched alkyl group having 1 to 6 carbon atoms, R 2 and R 3 are independently selected straight-chain or branched alkyl groups having 1 to 6 carbon atoms, or R 2 and R 3 together form a 4-membered, 5-membered, 6-membered, or 7-membered azacycloalkyl group having N to which they are attached as a heteroatom, and R 4 is hydrogen or halogen.) A further embodiment of the present invention is a specific method for tumor cells showing a combined effect of a compound (III) which is an antitumor agent or a pharmaceutically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, the method comprising simultaneously administering a specific combination of the compound (III) or a pharmaceutically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor to a plurality of tumor cells in vitro, and measuring the growth rate or cell viability of the plurality of tumor cells.

[0022] JPEG0007683931000008.jpg42170(wherein X is hydrogen, halogen, -NH 2is - CN, Y is a straight - chain or branched alkyl group having 1 to 6 carbon atoms, and Z 1 and Z 2 are each hydrogen or a halogen, and a straight - chain or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, where the substituent is hydroxy or phenyl, or Z 1 and Z 2 together form an azacycloalkyl group of a 4 - membered ring, 5 - membered ring, 6 - membered ring, or 7 - membered ring having N to which they are attached as a heteroatom. ) Said compound (III) may be compound (II).

[0023] JPEG0007683931000009.jpg44170(wherein R 5 is a straight - chain or branched alkyl group having 1 to 6 carbon atoms, R 2 and R 3 are independently selected straight - chain or branched alkyl groups having 1 to 6 carbon atoms, or R 2 and R 3 together form an azacycloalkyl group of a 4 - membered ring, 5 - membered ring, 6 - membered ring, or 7 - membered ring having N to which they are attached as a heteroatom, and R 4 is hydrogen or a halogen. ) In any of the above - mentioned specific methods, the tumor cells may be resistant to a glutathione - concentration - reducing agent or a glutathione S - transferase inhibitor. A further embodiment of the present invention is a radiation - combined antitumor agent for use in radiotherapy, which contains an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof as an active ingredient. JPEG0007683931000010.jpg44170(wherein R 5 is a straight - chain or branched alkyl group having 1 to 6 carbon atoms, R 2 and R 3 are independently selected straight - chain or branched alkyl groups having 1 to 6 carbon atoms, or R 2 and R 3 together form an azacycloalkyl group of a 4 - membered ring, 5 - membered ring, 6 - membered ring, or 7 - membered ring having N to which they are attached as a heteroatom, and R 4is hydrogen or a halogen.) The compound represented by the formula (II) may be oxyfedrine. It may be an antitumor agent against a tumor containing tumor cells resistant to a glutathione concentration reducing agent or a glutathione S-transferase inhibitor. Aldehyde dehydrogenase may be highly expressed in the tumor cells. The tumor may further contain tumor cells expressing CD44v. A further embodiment of the present invention is a method for measuring an antitumor effect, which comprises, in vitro, irradiating tumor cells with radiation in the presence of an aldehyde dehydrogenase inhibitor or a compound (III) or a pharmaceutically acceptable salt thereof, and measuring the growth rate or cell viability of the tumor cells. JPEG0007683931000011.jpg42170(wherein X is hydrogen, a halogen, -NH 2 , or -CN, Y is a linear or branched alkyl group having 1 to 6 carbon atoms, and Z 1 and Z 2 are each hydrogen or a halogen, and a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, the substituent being hydroxy or phenyl, or Z 1 and Z 2 together form a 4-membered, 5-membered, 6-membered, or 7-membered azacycloalkyl group having N to which they are attached as a heteroatom.) The tumor cells may be resistant to a glutathione concentration reducing agent or a glutathione S-transferase inhibitor. A further embodiment of the present invention is a method for identifying an agent having a synergistic effect with radiation irradiation in tumor cells in vitro, which comprises, in vitro, irradiating tumor cells with radiation in the presence of an aldehyde dehydrogenase inhibitor or each of a plurality of compounds (III) or a pharmaceutically acceptable salt thereof, and measuring the growth rate or cell viability of the tumor cells. JPEG0007683931000012.jpg42170(wherein X is hydrogen, a halogen, -NH 2 , or -CN, Y is a linear or branched alkyl group having 1 to 6 carbon atoms, and Z 1 and Z2 is each independently hydrogen or halogen, and a C1-6 linear or branched alkyl group which may have a substituent, wherein the substituent is hydroxy or phenyl, or Z 1 and Z 2 together form a 4-, 5-, 6- or 7-membered azacycloalkyl group having N to which they are attached as a heteroatom. ) The compound (III) may be the compound (II). JPEG0007683931000013.jpg44170(wherein R 5 is a C1-6 linear or branched alkyl group, R 2 and R 3 are independently selected C1-6 linear or branched alkyl groups, or R 2 and R 3 together form a 4-, 5-, 6- or 7-membered azacycloalkyl group having N to which they are attached as a heteroatom, and R 4 is hydrogen or halogen.) The tumor cells may be resistant to a glutathione concentration reducing agent or a glutathione S-transferase inhibitor. A further embodiment of the present invention is an enhancer of antitumor action by co-administration of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmaceutically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, which contains an inhibitor of the enhancing action of Nrf2 on xCT expression, and is an enhancer of antitumor action. JPEG0007683931000014.jpg44170(wherein R 5 is a C1-6 linear or branched alkyl group, R 6 is hydrogen or halogen, R 7 is a C1-6 linear or branched alkyl group which may have a substituent, wherein the substituent is hydroxy or phenyl, and R 8is hydrogen or a halogen. ) The inhibitor may be a substance that suppresses the expression of the Nrf2 gene or an inhibitor of Nrf2. The substance that suppresses the expression of the Nrf2 gene may be an antisense NA, miNA, or siNA against the Nrf2 gene. The inhibitor of Nrf2 may be ML385 or an anti-Nrf2 antibody. The antitumor effect may be an effect on a tumor overexpressing the Nrf2 gene. The glutathione concentration reducing agent may be sulfasalazine. The compound represented by the formula (II) may be oxyfedrine. A further embodiment of the present invention is a companion diagnostic agent for predicting an antitumor effect by co-administration of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmaceutically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, the companion diagnostic agent comprising a reagent for detecting Nrf2 gene expression. JPEG0007683931000015.jpg44170(wherein R 5 is a linear or branched alkyl group having 1 to 6 carbon atoms, R 6 is hydrogen or a halogen, R 7 is a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, the substituent being hydroxy or phenyl, R 8 is hydrogen or a halogen. ) The detection reagent may be an antibody, a gene expression detection probe, or a gene amplification primer. The glutathione concentration reducing agent may be sulfasalazine. The compound represented by the formula (II) may be oxyfedrine. A further embodiment of the present invention is a companion diagnostic agent for predicting an antitumor effect by co-administration of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmaceutically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, the companion diagnostic agent comprising a reagent for detecting a mutation in the Keap1 gene or the Nrf2 gene. JPEG0007683931000016.jpg44170(wherein R 5is a linear or branched C1-6 alkyl group, R 6 is hydrogen or halogen, R 7 is a linear or branched C1-6 alkyl group which may have a substituent, wherein the substituent is hydroxy or phenyl, R 8 is hydrogen or halogen.) ==Cross-reference with related documents== This application claims priority based on Japanese Patent Application No. 2019-091358 filed on May 14, 2019 and Japanese Patent Application No. 2019-200094 filed on November 1, 2019, and incorporates the said basic applications by reference herein.

Brief description of the drawings

[0024]

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Mode for Carrying Out the Invention

[0025] Hereinafter, embodiments of the present invention will be described in detail with reference to examples. It should be noted that the object, features, advantages, and ideas of the present invention are clear to those skilled in the art from the description in this specification, and those skilled in the art can easily reproduce the present invention from the description in this specification. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are presented for illustration or explanation purposes only, and do not limit the present invention thereto. It is obvious to those skilled in the art that various modifications and alterations can be made within the spirit and scope of the present invention disclosed in this specification based on the description in this specification.

[0026] In addition, when there is no particular explanation in the embodiments and examples, the methods described in standard protocol collections or methods modified or altered therefrom are used. Also, when using commercially available reagent kits or measuring devices, the protocols attached thereto are used unless otherwise specified.

[0027] ==Antitumor Agent== One embodiment of the present invention is an antitumor agent containing, as an active ingredient, a glutathione concentration reducing agent administered simultaneously with an effective amount of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmaceutically acceptable salt thereof. Here, the effective amount of the aldehyde dehydrogenase inhibitor is an amount of the aldehyde dehydrogenase inhibitor having a combined effect with the glutathione concentration reducing agent as an antitumor activity.

[0028] Another embodiment of the present invention is an antitumor agent containing, as an active ingredient, an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmaceutically acceptable salt thereof administered simultaneously with an effective amount of a glutathione concentration reducing agent. Here, the effective amount of the glutathione concentration reducing agent is an amount of the glutathione concentration reducing agent having a combined effect with the aldehyde dehydrogenase inhibitor as an antitumor activity.

[0029] Although not bound by the following theory, as shown in FIG. 12, in cells, there are multiple pathways for decomposing HNE, and it is considered that HNE accumulates in cells by simultaneously inhibiting two of these pathways, namely, the pathway via GST and the decomposition pathway via ALDH. And since HNE is cytotoxic, it is considered that tumor cells cannot proliferate. Therefore, by simultaneously administering an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmaceutically acceptable salt thereof and a glutathione concentration reducing agent, a synergistic effect is exhibited with respect to the antitumor effect.

[0030] In addition, another embodiment of the present invention is an antitumor agent containing, as an active ingredient, an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof, which is used in radiotherapy. Here, radiotherapy is a treatment method used for treating tumors, and its irradiation dose, irradiation method, etc. can be easily determined by a therapist based on common technical knowledge according to the state of the tumor and the patient. It is known that the amount of GSH in cells decreases upon radiation irradiation. By irradiating radiation in the presence of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof, a combined effect of both can be obtained. Therefore, in this case, an antitumor agent may be administered to a tumor patient, and radiation irradiation may be performed while a synergistic effect with radiation irradiation is observed at a concentration, or radiation may be irradiated to a tumor patient, and an antitumor agent may be administered while the amount of GSH is decreasing.

[0031] An aldehyde dehydrogenase inhibitor is a drug that inhibits the enzymatic activity of aldehyde dehydrogenase 2 (aldehyde dehydrogenase 2; ALDH) (EC 1.2.1.10). The type and isoform of ALDH to be inhibited are not particularly limited, and any of ALDH1 to 5 and their isoforms may be used. The aldehyde dehydrogenase inhibitor used as an antitumor agent is not particularly limited, but examples include chlorpropamide, tolbutamide, diethylaminobenzaldehyde, tetraethylthioperoxydicarbonic diamide, cyanamide, oxyfedrine, citral (3,7-dimethyl-2,6-octadienal), coprine, daidzin, DEAB (4-(Diethylamino)benzaldehyde), gossypol, kynurenine metabolites (3-hydroxykynurenine, 3-hydroxyanthranilic acid, kynurenic acid, and indol-3-ylpyruvic acid), molinate, nitroglycerin, pargyline (N-benzyl-N-methylprop-2-yn-1-amine) and their analogs, or their pharmacologically acceptable salts. In particular, dyclonine and dyclonine analog (I) shown below are preferred, and the compounds shown in Fig. 7 (BAS00363846, STL327701, PHAR033081, PHAR298639, and Aldi-2) are more preferred.

[0032] JPEG0007683931000017.jpg43170(wherein R 1 is a linear or branched alkyl group having 1 to 6 carbon atoms, and R 2 and R 3 are independently selected linear or branched alkyl groups having 1 to 6 carbon atoms, or R 2 and R 3 together form a 4-membered, 5-membered, 6-membered, or 7-membered azacycloalkyl group having N to which they are attached as a heteroatom, and R 4is hydrogen or a halogen. R 1 is preferably a linear or branched C4-5 alkyl group, R 2 and R 3 is a C2 alkyl group, or R 2 and R 3 preferably together form a 6-membered azacycloalkyl group having N to which they are attached as a heteroatom. Incidentally, R 1 is a linear C4 alkyl group, R 2 and R 3 preferably together form a 6-membered azacycloalkyl group having N to which they are attached as a heteroatom, and the compound is dichronine. The halogen is preferably F, Cl, I, Br, I.) Compound (II) is oxyfedrine or an analog thereof and has the following structural formula.

[0033] JPEG0007683931000018.jpg44170(wherein R 5 is a linear or branched C1-6 alkyl group, R 6 is hydrogen or a halogen, R 7 is a linear or branched C1-6 alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, R 8 is hydrogen or a halogen.) As the compound (II) to be used, oxyfedrine having the following structural formula (IV) is preferred, and as its salt, oxyfedrine hydrochloride is preferred.

[0034] JPEG0007683931000019.jpg49170 In addition, the pharmacologically acceptable salts are not limited as long as they can form salts with these compounds. Specifically, examples include addition salts of inorganic acids such as hydrochloride, sulfate, nitrate, hydrobromide, hydroiodide, perchlorate, and phosphate; addition salts of organic acids such as oxalate, maleate, fumarate, and succinate; addition salts of sulfonic acids such as methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, and camphorsulfonate; and addition salts of amino acids. Preferably, they are hydrochloride, oxalate, maleate, and methanesulfonate. Furthermore, it goes without saying that these compounds or their pharmacologically acceptable salts include not only anhydrides but also hydrates and crystal polymorphs.

[0035] A glutathione concentration-lowering agent is an agent that lowers the glutathione concentration in cells. The glutathione concentration-lowering agents used in these antitumor agents are not limited, but agents that inhibit the pathway by which glutathione is produced from cystine taken up into cells by xCT are preferred. More preferably, it is an agent that inhibits the activity of any one of xCT, Thioredoxin-1 (thioredoxin-1: TRX-1), glutamate-cysteine ligase (GCL) (EC6.3.2.2) (also called γ-glutamylcysteine synthetase), and glutathione synthetase (EC6.3.2.3). An xCT inhibitor or a GCL inhibitor is more preferred. The xCT inhibitor is not particularly limited, but preferably sulfasalazine, elastin, sorafenib, or their derivatives, or an anti-xCT antibody. The GCL inhibitor is also not particularly limited, but L-buthionine-sulfoximine or its derivatives are preferred. Here, the derivatives are not limited as long as they are glutathione concentration-lowering agents, and examples include PEGylated forms.

[0036] A glutathione S-transferase inhibitor is a drug that inhibits the enzymatic activity of glutathione S-transferase (EC 2.5.1.18), and in particular, a drug that inhibits the activity of converting HNE (4-HNE; 4-hydroxy-2-nonenal) to HNE-GSH. The glutathione S-transferase inhibitor is not particularly limited, and examples include glutathione analogs (for example, WO95 / 08563, WO96 / 40205, WO99 / 54346, etc.), ketoprofen, indomethacin, etacrynic acid, piroprostone, anti-GST antibodies, dominant negative mutants of GST, and the like.

[0037] In this specification, "administering two or more drugs simultaneously" means not only administering them simultaneously in time, but also administering each of them separately at different times as long as the other drug is administered while the effect of one drug remains. When administering two or more drugs simultaneously, two or more drugs containing one drug may be administered simultaneously, or two or more drugs may be formulated into one dosage form and administered. Note that "co-administration" and "co-administer" shall have the same meaning as "simultaneous administration" and "administer simultaneously", respectively.

[0038] The administration target of the anti-tumor agent is not particularly limited as long as it is a vertebrate, but it is preferably a human cancer patient. The tumor to be treated is not particularly limited, but a tumor containing tumor cells resistant to a glutathione concentration reducing agent or a glutathione S-transferase inhibitor is preferred. These tumor cells may have high expression of aldehyde dehydrogenase. The glutathione concentration reducing agent or the glutathione S-transferase inhibitor is preferably an xCT inhibitor, and more preferably sulfasalazine. Tumor cells with resistance refer to tumor cells that survive when administered to a patient at a normal treatment concentration for a normal treatment period in vivo, and in vitro, tumor cells with a survival rate of 90% or more at a concentration at which the survival rate is 50% or less in 80% or more of cell lines. For example, sulfasalazine-resistant tumor cells refer to those with an AUC in vivo when administered to a patient. 0-24Tumor cells that survive when administered at 50 to 300 μg·h / mL for about two weeks, and in vitro, can be defined as tumor cells with a survival rate of 90% or more at 200 μM. Also, L-buthionine-sulfoximine resistant cells, in vivo, are tumor cells that survive when administered to a patient with an AUC 0-24 of 10 to 100 μg·h / mL for about two weeks, and in vitro, can be defined as tumor cells with a survival rate of 90% or more at 100 μM. Sulfasalazine resistant tumor cells and L-buthionine-sulfoximine resistant cells preferably have low or negative CD44v expression. Tumor cells with overexpressed aldehyde dehydrogenase refer to cells in which the gene expression of any of ALDH1A1, ALDH2, ALDH1B1, or ALDH3A1 is expressed at a level 3 times or more, preferably 10 times or more higher than that of OSC19 cells. The tumor to be treated may contain tumor cells expressing CD44v. This is because sulfasalazine and L-buthionine-sulfoximine have an effective antitumor effect on tumor cells expressing CD44v. Tumor cells expressing CD44v may be any cells in which CD44v expression can be detected, but highly expressed cells are preferred. In that case, the high expression only needs to be the same as or higher than the average level of ovarian tumor cells, but preferably 2 times or more higher, more preferably 4 times or more higher, and even more preferably 10 times or more higher.

[0039] The type of tumor is not particularly limited, but solid cancer is preferred, and examples include colorectal adenocarcinoma, gastric adenocarcinoma, breast cancer, lung adenocarcinoma, pancreatic cancer, squamous cell carcinoma of the head and neck, ovarian tumor, and testicular tumor.

[0040] The antitumor agent may be formulated into tablets, powders, granules, powders, capsules, solutions, emulsions, suspensions, etc. by ordinary methods. In that case, it is manufactured using pharmaceutically acceptable additives known to those skilled in the art, such as excipients and carriers.

[0041] The anti-tumor agent may be administered to the subject to be administered in a suitable manner within the effective amount range. The effective amount can be finally determined as appropriate by the judgment of a physician or veterinarian in consideration of the type of dosage form, the administration method, the age and weight of the subject to be administered, the medical condition of the subject to be administered, and the like. For example, the dosage of the compound is preferably 0.1 mg / kg or more per day, more preferably 1 mg / kg or more, even more preferably 10 mg / kg or more, preferably 1000 mg / kg or less, more preferably 300 mg / kg or less, and even more preferably 100 mg / kg or less. The administration method is not particularly limited. For example, it may be administered orally, parenterally by injection or infusion into the abdominal cavity or vein, or directly into the cancer by injection or the like.

[0042] ==Enhancer of the anti-tumor effect of the anti-tumor agent== One embodiment of the present invention is an enhancer of the anti-tumor effect by co-administration of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, which contains an inhibitor of the xCT expression enhancing function of Nrf2. It can also be said to be an enhancer of the anti-tumor effect of the above-mentioned anti-tumor agent. This enhancer of the anti-tumor effect of the anti-tumor agent is administered simultaneously with the anti-tumor agent.

[0043] As shown in the examples, there is a positive correlation between the expression level of Nrf2 and the expression levels of xCT and ALDH. Without being bound by this theory, it is considered that suppressing the expression level of Nrf2 can suppress the expression of xCT and ALDH and enhance the effect of the anti-tumor agent.

[0044] Examples of the inhibitor of the xCT expression enhancing action of Nrf2 include an expression suppressing substance for the Nrf2 gene and an inhibitor of the xCT expression enhancing function of Nrf2. That is, in order to suppress the xCT expression enhancing action of Nrf2, the expression of the Nrf2 gene may be suppressed in tumor cells, or the xCT expression enhancing function as a protein of Nrf2 may be inhibited.

[0045] Examples of substances that suppress the expression of the Nrf2 gene include antisense NA, miNA, or siNA against the Nrf2 gene. Each of them may consist of RNA, DNA, or a chimeric molecule of RNA and DNA. Further, the nucleic acid (NA) may have various modifications. Their sequences can be easily designed from the common general knowledge of those skilled in the art. Also, examples of inhibitors of Nrf2 include low-molecular-weight compounds such as ML385 and anti-Nrf2 antibodies.

[0046] The tumor cells to be administered are not particularly limited, but tumors that overexpress the Nrf2 gene are preferred. This is because tumors that overexpress the Nrf2 gene are resistant to the above-mentioned antitumor agents. Therefore, before administering an enhancer of the antitumor action of the antitumor agent, the expression level of the Nrf2 gene in the tumor cells of the administration subject may be examined. When the expression level of the Nrf2 gene is normal, only the antitumor agent may be administered, or an enhancer of the antitumor action of the antitumor agent may also be administered simultaneously. When the expression level of the Nrf2 gene is higher than normal, it is preferable to co-administer the antitumor agent and an enhancer of the antitumor action of the antitumor agent.

[0047] ==Method for Measuring Proliferation Rate or Cell Survival Rate of Tumor Cells== One embodiment of the present invention is a measurement method including a step of simultaneously administering an aldehyde dehydrogenase inhibitor or compound (III) or a pharmaceutically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor to tumor cells in vitro, and a step of measuring the proliferation rate or cell survival rate of the tumor cells to which the drug has been administered. Another embodiment of the present invention is a measurement method including a step of irradiating tumor cells with radiation in vitro in the presence of an aldehyde dehydrogenase inhibitor or compound (III) or a pharmaceutically acceptable salt thereof, and a step of measuring the proliferation rate or cell survival rate of the tumor cells. The aldehyde dehydrogenase inhibitor, glutathione concentration reducing agent, and glutathione S-transferase inhibitor in this section shall be in accordance with those detailed in the section of "Antitumor Agents". Compound (III) has the following structural formula, and preferably it is Compound (II).

[0048] JPEG0007683931000020.jpg42170(Wherein, X is hydrogen, halogen, -NH 2 , or -CN, Y is a linear or branched alkyl group having 1 to 6 carbon atoms, and Z 1 and Z 2 are each hydrogen or halogen, and a linear or branched alkyl group having 1 to 6 carbon atoms which may have a substituent, the substituent being hydroxy or phenyl, or Z 1 and Z 2 together form a 4-membered, 5-membered, 6-membered, or 7-membered azacycloalkyl group having N to which they are attached as a heteroatom.) Since the aldehyde dehydrogenase inhibitor or Compound (III) or a pharmacologically acceptable salt thereof and the glutathione concentration reducing agent or glutathione S-transferase inhibitor have a combined effect on antitumor activity, by this measurement method, it is possible to find a combination of drugs with a high combined effect or to find particularly effective tumor cells for a combination of drugs.

[0049] Specifically, by simultaneously administering in vitro to tumor cells a specific glutathione concentration reducing agent or glutathione S-transferase inhibitor and each of a plurality of aldehyde dehydrogenase inhibitors or compound (III) or a pharmacologically acceptable salt thereof, and measuring the growth rate or cell viability of the tumor cells to which the drug has been administered, an aldehyde dehydrogenase inhibitor or compound (III) or a pharmacologically acceptable salt thereof having a combined effect with the specific glutathione concentration reducing agent or glutathione S-transferase inhibitor can be identified. Further, by simultaneously administering in vitro to tumor cells a specific aldehyde dehydrogenase inhibitor or compound (III) or a pharmacologically acceptable salt thereof and a plurality of glutathione concentration reducing agents or glutathione S-transferase inhibitors, and measuring the growth rate or cell viability of the tumor cells to which the drug has been administered, a glutathione concentration reducing agent or glutathione S-transferase inhibitor having a combined effect with the specific aldehyde dehydrogenase inhibitor or compound (III) or a pharmacologically acceptable salt thereof can be identified. Alternatively, in vitro, radiation is applied to tumor cells in the presence of an aldehyde dehydrogenase inhibitor or each of a plurality of compound (III) or a pharmacologically acceptable salt thereof, and by measuring the growth rate or cell viability of the tumor cells, a drug having a synergistic effect with radiation can be identified.

[0050] Furthermore, by simultaneously administering in vitro to a plurality of tumor cells a specific combination of an aldehyde dehydrogenase inhibitor or compound (III) or a pharmacologically acceptable salt thereof and a glutathione concentration reducing agent or glutathione S-transferase inhibitor, and measuring the growth rate or cell viability of the plurality of tumor cells to which the drug has been administered, tumor cells showing a combined effect of the aldehyde dehydrogenase inhibitor or compound (III) or a pharmacologically acceptable salt thereof and the glutathione concentration reducing agent or glutathione S-transferase inhibitor can be identified. The compound (III) used in these methods is preferably an antitumor agent having antitumor activity.

[0051] ==Companion diagnostic agent== One embodiment of the present invention is a companion diagnostic agent for predicting the antitumor effect of the above-described antitumor agent, which comprises a reagent for detecting Nrf2 gene expression. In recent years, it has become known that the expression of Nrf2 is a tumor malignancy factor. As shown in the examples, tumor cells with a high expression level of Nrf2 have a relatively weak effect of the antitumor agent. Without being bound by this theory, there is a positive correlation between the expression level of Nrf2 and the expression levels of xCT and ALDH, and since the above-described antitumor agent simultaneously suppresses the expression of xCT and ALDH, it is considered that when the expression level of Nrf2 is high, it becomes difficult to exert the effect of the antitumor agent. Therefore, it is expected that the higher the expression level of the Nrf2 gene, the weaker the effect of the antitumor agent, and the lower the expression level of the Nrf2 gene, the stronger the effect of the antitumor agent. The detection of Nrf2 gene expression can be performed at any stage leading to the final product of the Nrf2 protein. For example, mRNA or protein may be detected. The reagent for detecting Nrf2 gene expression is not particularly limited and can be easily selected from common general knowledge. For example, it may include an antibody, a probe for detecting gene expression, or a primer for gene amplification. Those skilled in the art can easily produce an anti-Nrf2 antibody and easily design a probe for detecting gene expression or a primer for gene amplification from common general knowledge. In addition, since the constitutive expression of the Nrf2 protein may be related to mutations in the Keap1 and Nrf2 genes, the companion diagnostic agent is also a reagent for detecting known mutations in the Keap1 gene or Nrf2 gene. The detection of mutations in the Keap1 gene or Nrf2 gene can be performed by known techniques. For example, it may include a primer for gene amplification for amplifying the Keap1 gene or Nrf2 gene.

Example

[0052] (Experimental Example 1) Combined effect of sulfasalazine and dyclonine (Objective) In this experimental example, it is shown that sulfasalazine and dyclonine, both of which have an xCT inhibitory effect, exhibit a combined effect on reducing the viability of sulfasalazine-resistant cells.

[0053] (Method) 2000 oral squamous cell carcinoma cell line HSC-4, a sulfasalazine-resistant cell line, were seeded per well in a 96-well plate and culture was initiated. DMEM was used as the medium. After 24 hours, the medium was replaced with a medium containing 50 μM dyclonine or an equal volume of DMSO and 0 μM (no addition), 50 μM, 100 μM, 200 μM, or 400 μM sulfasalazine, and culture was continued for 48 hours. Subsequently, cell viability was measured using Celltiter-Glo (Promega), and the cell viability in each case was calculated with the number of viable cells in the control (DMSO added, sulfasalazine not added) set as 100%. A graph showing the viability against each concentration of sulfasalazine was created in Fig. 1.

[0054] (Results) HSC4 is a sulfasalazine-resistant cell line, and sulfasalazine alone has little effect on cell viability. Also, dyclonine alone (dyclonine added, sulfasalazine not added) shows a viability of 80%. However, when both dyclonine and sulfasalazine are added, the viability becomes 10% or less when sulfasalazine is 100 μM or more.

[0055] Thus, sulfasalazine and dyclonine exhibit a combined effect on reducing the viability of sulfasalazine-resistant cells.

[0056] (Experimental Example 2) Change in dyclonine sensitivity by xCT knockdown (Objective) In this experimental example, by showing that a similar combined effect with dyclonine can be obtained even when xCT is knocked down instead of sulfasalazine, which has an xCT inhibitory effect, it is shown that the combined effect of sulfasalazine and dyclonine is mediated through the xCT inhibitory effect of sulfasalazine.

[0057] (Method) 3000 oral squamous cell carcinoma cell line HSC-4 cells, which are sulfasalazine-resistant cell lines, were seeded in a 96-well plate at 3000 cells / well, and non-silencing control (scramble (Sense: UUCUCCGAACGUGUCACGUtt (SEQ ID NO: 1), Antisense: ACGUGACACGUUCGGAGAAtt (SEQ ID NO: 2))) siRNA or xCT-specific siRNA (xCT siRNA#1 Sense: AGAAAUCUGGAGGUCAUUAtt (SEQ ID NO: 3), Antisense: AGAAAUCUGGAGGUCAUUAtt (SEQ ID NO: 4), xCT siRNA#2 Sense: CCAGAACAUUACAAAUAAUtt (SEQ ID NO: 5), Antisense: AUUAUUUGUAAUGUUCUGGtt (SEQ ID NO: 6)) was lipofected using Lipofectamine RNAiMAX (ThermoFisher Scientific), and the culture was started. DMEM was used as the medium. After 24 hours, the medium was replaced with a medium containing 50 μM diclofenac (the solvent was DMSO) or an equal volume of DMSO, and the culture was continued for 48 hours. Then, Celltiter-Glo (Promega) was used to measure the cell viability, and the cell viability of the control (non-silencing control, DMSO added) was set as 100%, and the cell viability of each group was calculated. The results are shown in Figure 2.

[0058] (Results) HSC-4 has a cell viability of about 60% with 50 μM diclofenac alone, while when xCT is knocked down, it has a cell viability of only about 10 - 20% in the presence of 50 μM diclofenac.

[0059] Thus, the combined effect of sulfasalazine and diclofenac is mediated through the xCT inhibitory effect of sulfasalazine.

[0060] (Experimental Example 3) Combined effects of sulfasalazine, elastin, or BSO and diclofenac in various cancer cell lines (Objective) In this experimental example, it is shown that sulfasalazine, elastin which is a specific inhibitor of xCT, or BSO which is a glutathione synthesis inhibitor and diclofenac have a combined effect in various tumor cell lines, and at the same time, it is shown that the inhibition of xCT is mediated through the inhibition of glutathione synthesis.

[0061] (Method) The cell lines shown in Figure 3 were seeded at 3000 cells / well in a 96-well plate and culture was started. DMEM was used as the medium. After 24 hours, the medium was replaced with a medium containing 50 μM diclofenac or an equal amount of DMSO, and 0 μM (no addition) or 400 μM sulfasalazine, 0 μM (no addition) or 5 μM elastin, 0 μM (no addition) or 100 μM BSO, and culture was continued for 48 hours. Then, the cell viability was measured using Celltiter-Glo (Promega), and the cell viability of each was calculated with the number of viable cells in the control (DMSO added, diclofenac not added) set as 100%. The cell viability in each case was illustrated in Figure 3.

[0062] (Result) Although there were differences depending on the cells, a similar combined effect was observed in any combination of sulfasalazine, elastin, or BSO and diclofenac.

[0063] Thus, the inhibition of xCT by sulfasalazine or elastin exerts its antitumor effect through the inhibition of glutathione synthesis. Therefore, a glutathione concentration reducing agent or a glutathione S-transferase inhibitor can be used instead of sulfasalazine or elastin.

[0064] (Experimental Example 4) Combined effect of sulfasalazine and diclofenac in vivo (Objective) In this experimental example, it is shown that the combined effect of sulfasalazine and diclofenac is also observed in vivo.

[0065] (Method) 1×10 of the oral squamous cell carcinoma cell line HSC-2 cells, which are sulfasalazine-resistant cell lines 6Individuals were transplanted subcutaneously into nude mice, and from the 4th day after transplantation, physiological saline, sulfasalazine alone, dyclonine alone, and both sulfasalazine and dyclonine were intraperitoneally administered once a day at a dose of 400 mg / kg of sulfasalazine and 5 mg / kg of dyclonine, and this was continued until the 22nd day. Every 3 - 4 days, the short diameter and long diameter of the tumor were measured, the tumor volume was calculated using the following formula, and the results were graphed in Figure 4.

[0066] Tumor volume = (long diameter × (short diameter) 2 ) / 2 In addition, the statistical analysis of the tumor volume was performed by t - test on the 22nd day.

[0067] (Results) As shown in Figure 4, when each drug was administered alone, the tumor volume decreased by about 35%, but when both were administered, the volume decreased by about 70%.

[0068] Thus, the combined administration of sulfasalazine and dyclonine can reduce the growth of sulfasalazine - resistant tumors.

[0069] (Experimental Example 5) Inhibition of ALDH by Dyclonine (Objective) In this experimental example, it is shown that dyclonine has inhibitory activity against ALDH.

[0070] (Method) The oral squamous cell carcinoma cell line HSC - 4 cells were seeded at 8×10 in a 10 - cm cell culture dish. 5Seeding was carried out at a density of cells per dish, and the culture was initiated. DMEM was used as the culture medium. After 24 hours, the medium was replaced with a medium containing 50 μM of dyclonine (the solvent was DMSO), and the cells were cultured for 24 hours. Thereafter, the cells were collected, and cells having ALDH activity in the presence of N,N-diethylaminobenzaldehyde (DEAB) were stained using an ALDEFLUOR kit (STEMCELL Technologies) and analyzed by FACS (Dyclonine in the figure). As a control, the experimental results for those that were not stained with the ALDEFLUOR kit without adding DEAB (Unstained in the figure) and those that were stained with the ALDEFLUOR kit after replacing the medium with an equal amount of DMSO not containing dyclonine (Non-treatment in the figure) are shown. For the measurement of positive cells, a gate was created such that the percentage of positive cells was almost 0% for the DMSO-treated sample stained with the ALDEFLUOR kit in the presence of DEAB (DEAB in the figure), and the positive rate in each case was calculated.

[0071] (Results) As shown in Fig. 5, in the DMSO-treated cells, a cell population with high ALDH activity exists at about 25%, but in the dyclonine-treated cells and the DEAB-treated cells which are a known ALDH inhibitor, the cell population with high ALDH activity is suppressed to about 1%.

[0072] Thus, dyclonine has an inhibitory activity against ALDH.

[0073] (Experimental Example 6) Accumulation of HNE by the combined use of sulfasalazine and dyclonine (Objective) In this experimental example, it is shown that the combined use of sulfasalazine and dyclonine significantly increases the level of HNE in tumor cells and the frequency of cells that accumulate HNE.

[0074] (Method) Similar to Experimental Example 1, HSC-4 cells were cultured in a medium containing 50 μM diclofenine or an equal amount of DMSO and 0 μM (no addition) or 400 μM sulfasalazine, and the treated cells were fixed with 4% PFA-PBS. Further, after performing cell membrane permeabilization treatment with 0.2% Triton X-100-PBS, blocking was performed with 3% BSA-PBS. Then, fluorescence staining was performed using an anti-HNE antibody as the primary antibody and an Alexafluor488-labeled anti-mouse IgG antibody as the secondary antibody. As a positive control, cells incubated with 50 μM HNE for 30 minutes were used, and antibody staining was performed in the same manner. The observation image under a fluorescence microscope is shown in Fig. 6.

[0075] (Result) When diclofenine or sulfasalazine was treated alone, an increase in intracellular HNE concentration was observed at a low frequency. However, when sulfasalazine and diclofenine were used in combination, accumulation of high-concentration intracellular HNE was observed at a high frequency.

[0076] Thus, by using an xCT inhibitor and an ALDH inhibitor in combination, accumulation of high-concentration intracellular HNE at a high frequency can be observed. For this reason, as described above, there are multiple pathways for decomposing HNE in cells (see Fig. 12). By simultaneously inhibiting two of these pathways, namely the pathway via GST and the degradation pathway via ALDH, it is considered that HNE accumulates in cells. And since HNE is cytotoxic, it is considered that tumor cells cannot proliferate.

[0077] (Experimental Example 7) Combined effect of sulfasalazine or BSO and diclofenine analogs (having a diclofenine skeleton) (Objective) The following diclofenine analog (I) having a diclofenine skeleton is shown to have a combined effect with sulfasalazine or BSO.

[0078] JPEG0007683931000021.jpg43170(wherein, R 1 is a linear or branched alkyl group having 1 to 6 carbon atoms, R 2 and R 3is a C1-6 linear or branched alkyl group independently selected, or R 2 and R 3 together form a 4-membered, 5-membered, 6-membered, or 7-membered azacycloalkyl group having as a heteroatom the N to which they are attached, and R 4 is hydrogen or halogen. R 1 is preferably a C4-5 linear or branched alkyl group, and R 2 and R 3 are a C2 alkyl group, or preferably R 2 and R 3 together form a 6-membered azacycloalkyl group having as a heteroatom the N to which they are attached. Note that R 1 is a C4 linear alkyl group, and the compound in which R 2 and R 3 together form a 6-membered azacycloalkyl group having as a heteroatom the N to which they are attached is dichlorin. Halogen is preferably F, Cl, I, Br, I.) (Method) Similar to Experimental Example 1, HSC-4 cells were cultured in a medium containing 0 μM (no addition), 25 μM, 50 μM, or 100 μM of dichlorin, or 12.5 μM, 25 μM, 50 μM, 100 μM of dichlorin analog BAS00363846, STL327701, PHAR033081, PHAR298639, or Aldi-2 (see Structural Formula in Figure 7B), and 0 μM (no addition) or 100 μM of BSO or 300 μM of sulfasalazine, and the cell viability was measured and graphed in Figure 7A.

[0079] (Result) All of these compounds showed a combined effect with BSO or sulfasalazine.

[0080] Thus, the dichlorin analog (I) having a dichlorin skeleton has a combined effect as an xCT inhibitor and an antitumor agent.

[0081] (Experimental Example 8) Combined effect of BSO and dichlorin analog (without dichlorin skeleton) (Purpose) To show that a dichlorin analog without a dichlorin skeleton has no combined effect with BSO.

[0082] (Method) Similar to Experimental Example 1, HSC-4 cells were cultured in a medium containing 0 μM (no addition), 12.5 μM, 25 μM, or 50 μM dichlorine, or 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM dichlorine analog (4-hydroxyacetophenone: see Structural Formula in Figure 8B), and 0 μM (no addition) or 100 μM BSO, and the cell viability was measured and graphed in Figure 8A.

[0083] (Result) Dichlorine analogs without the dichlorine skeleton did not show a combined effect with BSO.

[0084] Thus, the dichlorine skeleton is important for the interaction with xCT inhibitors.

[0085] (Experimental Example 9) Combined effect of sulfasalazine, elastin, or BSO and dichlorine in sulfasalazine-resistant OSC19 cells (Objective) To show that dichlorine has a combined effect with a glutathione synthesis inhibitor in cancer cell lines that have acquired resistance to xCT inhibitors.

[0086] (Method) The sulfasalazine-sensitive oral squamous cell carcinoma cell line OSC19 was cultured in DMEM medium containing sulfasalazine for 2 months to establish sulfasalazine-resistant OSC19 cells. The parental strain of OSC19 cells or OSC19-SSZR cells were seeded at 3000 cells / well in a 96-well plate. After 24 hours of culture, the medium was replaced with a medium containing sulfasalazine, elastin, or BSO at the concentrations shown in Figure 9, and 50 μM dichlorine (solvent: DMSO) or an equal amount of DMSO, and cultured for 48 hours. Then, the cell viability was measured using Celltiter-Glo (Promega), and the cell viability was calculated with the control (no addition of sulfasalazine, elastin, and BSO, addition of DMSO) set as 100%.

[0087] (Result) Dyclonine also showed a combined effect with sulfasalazine, elastin, or BSO in OSC19-SSZR cells.

[0088] Thus, dyclonine also shows a combined effect with a glutathione synthesis inhibitor even in cancer cells that have acquired resistance to xCT inhibitors.

[0089] (Experimental Example 10) Expression of ALDH gene family in sulfasalazine-resistant OSC19 cells and HSC-4 (Objective) To show that the ALDH gene family is highly expressed in cancer cells resistant to xCT inhibitors.

[0090] (Method) Messenger RNA was extracted from HSC-4 cells, OSC19 cells, and OSC19-SSZR cells, and complementary DNA was synthesized by performing a reverse transcription reaction. Then, using the obtained complementary DNA as a template, the expression levels of ALDHIAl, ALDHIBl, ALDH2, ALDH3Al, and RPS17 were measured by quantitative RT-PCR. Using the expression level of RPS17 as a reference, the expression levels of each ALDH family gene were quantified by the ΔΔCt method and graphed in Figure 10.

[0091] (Result) The expression of ALDHIAl was increased in OSC19-SSZR compared to OSC19. High expression of ALDHIBl and ALDH2 was observed in HSC-4. ALDH3Al was highly expressed in HSC4 and OSC19-SSZR. Thus, the expression of the ALDH family genes tended to be high in xCT-insensitive cancer cell lines.

[0092] ​Thus, in cancer cells with high expression of ALDH family genes, since HNE is decomposed by the ALDH family genes, even if the decomposition into GST is suppressed by an xCT inhibitor, the toxicity of HNE does not act, and resistance to the xCT inhibitor is obtained (see Fig. 12). When an ALDH inhibitor is administered to such cells, the sensitivity to the xCT inhibitor increases. Therefore, an antitumor agent containing an ALDH inhibitor and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor effectively acts on cancer cells with high expression of ALDH family genes.

[0093] (Experimental Example 11) Combined effect of oxyfedrine (OXY) and sulfasalazine (SSZ) or L-buthionine-sulfoximine (BSO) (Objective) In this experimental example, it is shown that sulfasalazine or L-buthionine-sulfoximine and oxyfedrine have a combined effect on the reduction of the survival rate of sulfasalazine or L-buthionine-sulfoximine-resistant tumor cells (A549, HCT116, HSC-4).

[0094] (Method) 4,000 cells / well of the alveolar basal epithelial adenocarcinoma cell line A549, the colon adenocarcinoma cell line HCT116, and the oral squamous cell carcinoma cell line HSC-4, which are sulfasalazine or L-buthionine-sulfoximine-resistant cell lines, were seeded in a 96-well plate and culture was started. RPMI was used for A549 and DMEM was used for HCT116 and HSC-4 as the medium. After 24 hours, the medium was replaced with a medium containing 50 μM or 100 μM oxyfedrine or an equal amount of DMSO, 0 μM (no addition of either sulfasalazine or L-buthionine sulfoximine), 400 μM sulfasalazine, or 100 μM L-buthionine sulfoximine, and the culture was continued for 48 hours. Then, the cell viability was measured using Celltiter-Glo (Promega), and the cell survival rate in each case was calculated with the number of viable cells in the control (addition of DMSO, no addition of either sulfasalazine or L-buthionine sulfoximine) set as 100%. A graph showing the survival rate for each concentration of oxyfedrine was created in Fig. 11.

[0095] (Result) A549, HCT116, and HSC4 are sulfasalazine- and / or buthionine sulfoximine-resistant cell lines, and sulfasalazine or buthionine sulfoximine alone has little effect on cell viability. Also, at 50 μM of oxyfedrine alone (oxyfedrine added, sulfasalazine not added), no significant cell-killing effect was observed. However, when both oxyfedrine and sulfasalazine or buthionine sulfoximine are added, for example, when oxyfedrine is 100 μM, the survival rate is 25% or less under combination with sulfasalazine and 5% or less under combination with buthionine sulfoximine.

[0096] Thus, regarding the decrease in the survival rate of sulfasalazine-resistant cells, sulfasalazine or buthionine sulfoximine and oxyfedrine have a synergistic combined effect. On the other hand, this effect varies somewhat depending on the cell. For example, considering administration to patients, a low concentration is preferable. Regarding the combined effect of oxyfedrine at 50 μM with sulfasalazine, in A549 cells, it is almost the same as the cell survival rate with sulfasalazine alone, but in HCT116 cells, the cell survival rate is 75%, and also in HSC4 cells, it is 75%. The combined effect of oxyfedrine and sulfasalazine is weaker in A549 than in HCT116 cells and HSC-4 cells.

[0097] (Experimental Example 12) Decrease in intracellular GSH concentration in tumor cells by sulfasalazine (SSZ) or buthionine sulfoximine (BSO) (Objective) In this experimental example, it is shown that the intracellular GSH concentration decreases by SSZ or BSO.

[0098] (Method) Using SSZ-resistant tumor cells (HCT116, HSC-4), the cells were treated in the same manner as in Example 11, and the intracellular GSH concentration after 48 hours was measured using a GSH-Glo glutathione assay kit (Promega). The measurement results are shown in Fig. 13.

[0099] (Results) When SSZ, BSO, or oxyfedrine (OXY) was treated alone, the intracellular GSH concentration decreased with SSZ or BSO alone, but the effect on the decrease in intracellular GSH concentration with SSZ alone was not very significant. And with OXY alone, the intracellular GSH concentration did not decrease. On the other hand, when SSZ and OXY were used in combination, a decrease in intracellular GSH concentration was observed compared to SSZ alone.

[0100] Thus, SSZ or BSO functions as an xCT inhibitor and decreases the GSH concentration in tumor cells, but the effect is not sufficient with SSZ alone.

[0101] (Experimental Example 13) Accumulation of HNE by the combined use of sulfasalazine (SSZ) or buthionine sulfoximine (BSO) and oxyfedrine (OXY) (Objective) In this experimental example, it is shown that the combined use of SSZ or BSO and OXY significantly increases the level of HNE in tumor cells and the frequency of cells accumulating HNE.

[0102] (Method) In this experimental example, sulfasalazine-resistant tumor cells (HCT116, HSC-4) were used, and the cells were treated in the same manner as in Experimental Example 6 and observed under a fluorescence microscope. The observation images under the fluorescence microscope are shown in Fig. 14.

[0103] (Results) When SSZ, BSO, or OXY was treated alone, cells with an increase in intracellular HNE concentration were observed at a low frequency, but when SSZ or BSO was combined with OXY, cells with a high frequency and high concentration of intracellular HNE accumulation were observed.

[0104] Thus, by combining an xCT inhibitor and an ALDH inhibitor, cells with a high frequency and high concentration of intracellular HNE accumulation could be observed. Accordingly, regarding the decrease in the survival rate of sulfasalazine-resistant cells as in Example 11, it is considered that the xCT inhibitor and the ALDH inhibitor have a synergistic combined effect.

[0105] (Experimental Example 14) Combined effect of sulfasalazine (SSZ) and oxyphedrine (OXY) in vivo (Objective) This experimental example shows that the combined effect of SSZ and OXY can also be observed in vivo.

[0106] (Method) Using the oral squamous cell carcinoma cell line HCT-116 cells, which are SSZ-resistant cell lines, tumors were formed in mouse bodies in the same manner as in Example 4, and the tumor volume (7 days and 14 days after transplantation) and tumor weight (16 days after transplantation) were calculated, and the results were graphed in Figure 15. The photographs of the tumors were taken at the time of measuring the tumor weight.

[0107] (Result) As shown in Figure 15, when each drug was used alone, there was almost no tumor growth inhibitory effect, and the growth of the tumor was significantly inhibited by the combination of SSZ and OXY.

[0108] Thus, the combined administration of SSZ and OXY can suppress the growth of tumors derived from SSZ-resistant cells.

[0109] (Experimental Example 15) Accumulation of HNE in in vivo tumors by the combination of sulfasalazine (SSZ) and oxyphedrine (OXY) (Objective) This experimental example shows that the combined use of SSZ and OXY significantly increases the level of HNE and the frequency of cells accumulating HNE in tumors formed in vivo.

[0110] (Method) Using the oral squamous cell carcinoma cell line HCT-116 cells, which are SSZ-resistant cell lines, tumors were formed in mice in the same manner as in Example 4. Immunohistochemical analysis was performed on the tumors 16 days after transplantation as follows. First, the tumor tissue was fixed with a 4% formaldehyde solution, and paraffin sections were prepared. After performing cell membrane permeabilization treatment with 0.2% Triton X100-PBS, it was washed with PBS and blocked with 3% BSA-PBS. Then, using the Vectastain Elite kit (Vector Laboratories), an anti-HNE antibody was used as the primary antibody, and ImmPACT DAB Peroxidase Substrate (Vector Laboratories) was used as the enzyme substrate to stain HNE brown. The observation image under the microscope is shown in Fig. 16.

[0111] (Result) As shown in Fig. 16, for in vivo tumors, by combining SSZ and OXY, cells with a high frequency and high concentration of intracellular HNE accumulation could be observed. Thus, it is considered that SSZ and OXY have a synergistic combined effect on the growth inhibition of tumors derived from SSZ-resistant cells, as in Example 14.

[0112] Thus, regarding the growth inhibition of tumors derived from sulfasalazine-resistant cells, the xCT inhibitor and the ALDH inhibitor have a synergistic combined effect.

[0113] (Experimental Example 16) Effect of using an ALDH inhibitor in the treatment of tumors by radiation (Objective) In this experimental example, by irradiating sulfasalazine (SSZ)-resistant cells with radiation and simultaneously using an ALDH inhibitor, it is shown that radiation and the ALDH inhibitor have a synergistic effect on the reduction of the survival rate of SSZ-resistant cells and the accumulation of HNE.

[0114] (Method) In this experimental example, SSZ-resistant tumor cells (HCT116, HSC-4) were irradiated with 4, 6, and 10 Gy of ionizing radiation using an X-ray irradiation device (Hitachi MBR-1520R-4, settings: 150 kV, 20 mA) in the presence of 50 μM oxyfedrine (OXY). As controls, samples without OXY and samples not irradiated with ionizing radiation (0 Gy) were also processed simultaneously. After 24 hours, the cell viability was calculated in the same manner as in Experimental Example 1. The results are shown in Fig. 17. Also, intracellular HNE was visualized in the same manner as in Example 6. The results are shown in Fig. 18.

[0115] (Result) As shown in Fig. 17, when irradiated with radiation in the presence of OXY, the cell viability decreased significantly and remarkably compared to the cases of radiation irradiation or OXY treatment alone. Also, as shown in Fig. 18, high-frequency and high-concentration intracellular HNE-accumulating cells were observed by the combined use of irradiation treatment and OXY treatment.

[0116] Thus, it is considered that the combined use of radiation irradiation and OXY treatment causes a high-concentration accumulation of intracellular HNE, thereby significantly reducing the cell viability. (Experimental Example 17) Correlation between the expression level of Nrf2 and the expression levels of xCT and ALDH (Objective) In this experimental example, it is shown that the expression level of Nrf2 has a positive correlation with the expression levels of xCT and ALDH.

[0117] (Method) The expressions of Nrf2, xCT, and β-actin were detected by Western blotting using primary antibodies against Nrf2, xCT, and β-actin and HRP-conjugated secondary antibodies for extracts of SSZ-resistant tumor cells (HCT116, HSC-4, A549). Chemiluminescence reagent Plus (Perkin-Elmer Japan) was used for detection. The results are shown in Fig. 19(A). Next, siRNA against the Nrf2 gene was lipofected into A549 cells to suppress the expression of the Nrf2 gene. After 48 hours, extracts of the lipofected A549 cells were obtained and Western blotting was performed in the same manner to examine the expression of Nrf2, xCT, ALDH3A1, and β-actin. The following sequences were used for the siRNA (the bases shown in lowercase are DNA overhangs). Control: 5'- UUCUCCGAACGUGUCACGUtt -3' (SEQ ID NO: 7) 5'- ACGUGACACGUUCGGAGAAtt -3' (SEQ ID NO: 8) siNrf2: 5'- UCCUACUGUGAUGUGAAAUtt -3' (SEQ ID NO: 9) 5'- AUUUCACAUCACAGUAGGAgc -3' (SEQ ID NO: 10) The results are shown in Fig. 19(B).

[0118] (Results) As shown in Fig. 19(A), in A549 cells, Nrf2 was overexpressed, and accordingly, xCT was overexpressed. Then, when the expression of the Nrf2 gene was suppressed by siRNA in A549 cells, the expression of xCT and ALDH3A1 was also suppressed.

[0119] Thus, the expression level of the Nrf2 gene is positively correlated with the expression levels of xCT and ALDH. In particular, in A549 cells, the Nrf2 gene is overexpressed.

[0120] (Experimental Example 18) Combined effects of siRNA against the Nrf2 gene, sulfasalazine (SSZ) or buthionine sulfoximine (BSO), and oxyphedrine (OXY) (Objective) From Fig. 11, when comparing the results of A549 with those of HCT116 or HSC4, the combined effect of oxyphedrine and sulfasalazine is weaker in A549. In this experimental example, it is shown that by suppressing the Nrf2 gene, the combined effect in A549 is enhanced.

[0121] (Method) For A549 cells, siRNA against the Nrf2 gene was introduced in the same manner as in Example 17, or the medium was supplemented with ML385, an inhibitor of Nrf2. Then, in the same manner as in Example 11, after culturing for 48 hours in a medium containing 50 μM of OXY and 400 μM of SSZ or 100 μM of BSO, the cell viability was measured. The results are shown in Figure 20. As a control, experiments were conducted in a medium without any additives, but DMSO was appropriately added so that the amount of DMSO added was constant in all cases.

[0122] (Result) From Figure 20, it can be seen that in A549 cells, in addition to SSZ + OXY, the cell viability can be effectively decreased by siRNA against the Nrf2 gene or ML385, an inhibitor of Nrf2.

[0123] Thus, the expression of the Nrf2 gene can be an indicator of whether the co - administration of SSZ + OXY is effective in suppressing tumor growth. And by further administering siRNA against the Nrf2 gene or ML385 in addition to SSZ + OXY, the effect of SSZ + OXY can be enhanced. This strategy is particularly effective against tumors with over - expressed Nrf2 gene. (Example 19) Effect of using an ALDH inhibitor in the treatment of tumors by radiation (Objective) In this experimental example, by irradiating tumors transplanted into nude mice with radiation and using an ALDH inhibitor, it is shown that radiation and the ALDH inhibitor have a synergistic effect on the decrease in viability of SSZ - resistant cells and the accumulation of HNE. (Method) 1.5×10 of the oral squamous cell carcinoma cell line HSC - 2, a sulfasalazine - resistant cell line 6They were subcutaneously transplanted into nude mice (5 mice). From the day after transplantation for 3 days (Day1 - Day3), oxyfedrine (OXY) (20 mg / kg) was intraperitoneally administered. On Day4, oxyfedrine (30 mg / kg) was intraperitoneally administered, and 2 hours later, X-rays (4, 6, or 10 Gy) were irradiated to the nude mice. Thereafter, for another 3 days (Day5 - Day7), oxyfedrine (20 mg / kg) was intraperitoneally administered, and then on Day7, the tumors were collected and their weights were measured. The results were shown in a bar graph in Figure 21. In addition, for the nude mice transplanted in the same way, the same oxyfedrine administration was performed, and the groups without X-ray irradiation and the groups without oxyfedrine administration but with the same X-ray irradiation were used as controls. Also, for statistical processing, for each X-ray irradiation dose, a Student's t-test was performed between the oxyfedrine-administered group and the non-administered group, and p < 0.05 was considered significant. (Results) As shown in Figure 21, as the X-ray intensity increased, the increase in tumor weight was suppressed. And in the group without X-ray irradiation and the group irradiated with 4 Gy X-rays, compared with the non-oxyfedrine-administered group, the oxyfedrine-administered group tended to have a more suppressed increase in tumor weight. However, in the group irradiated with 6 Gy X-rays and the group irradiated with 10 Gy X-rays, compared with the non-oxyfedrine-administered group, the oxyfedrine-administered group significantly suppressed the increase in tumor weight. Thus, radiation irradiation and oxyfedrine administration have a synergistic effect on suppressing the increase in tumor weight in vivo.

Industrial Applicability

[0124] According to the present invention, it has become possible to provide a novel antitumor agent and a formulation.

Claims

1. An antitumor agent containing, as an active ingredient, a glutathione concentration reducing agent or a glutathione S-transferase inhibitor administered simultaneously with an effective amount of oxyfedrine or a pharmacologically acceptable salt thereof, wherein the glutathione concentration reducing agent is selected from sulfasalazine, elastin, sorafenib, anti-xCT antibody, and L-buthionine-sulfoximine and its PEGylated forms, and the glutathione S-transferase inhibitor is selected from glutathione analogs, ketoprofen, indomethacin, etacrynic acid, piroprofen, anti-GST antibody, and dominant negative mutants of GST, an antitumor agent.

2. An antitumor agent containing, as an active ingredient, oxyfedrine or a pharmacologically acceptable salt thereof administered simultaneously with an effective amount of a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, wherein the glutathione concentration reducing agent is selected from sulfasalazine, elastin, sorafenib, anti-xCT antibody, and L-buthionine-sulfoximine and its PEGylated forms, and the glutathione S-transferase inhibitor is selected from glutathione analogs, ketoprofen, indomethacin, etacrynic acid, piroprofen, anti-GST antibody, and dominant negative mutants of GST, an antitumor agent.

3. The antitumor agent according to claim 2, wherein the glutathione concentration reducing agent is sulfasalazine or L-buthionine-sulfoximine.

4. A formulation containing, as active ingredients, oxyfedrine or a pharmacologically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, wherein the formulation is an antitumor agent, the glutathione concentration reducing agent is selected from sulfasalazine, elastin, sorafenib, anti-xCT antibody, and L-buthionine-sulfoximine and its PEGylated forms, and the glutathione S-transferase inhibitor is selected from glutathione analogs, ketoprofen, indomethacin, etacrynic acid, piroprofen, anti-GST antibody, and dominant negative mutants of GST, a formulation.

5. The formulation according to claim 4, wherein the glutathione concentration reducing agent is sulfasalazine or L-buthionine-sulfoximine.

6. An antitumor agent containing the formulation according to claim 4 or 5.

7. An antitumor agent against a tumor containing tumor cells resistant to a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, wherein the glutathione concentration reducing agent is selected from sulfasalazine, elastin, sorafenib, anti-xCT antibody, and L-buthionine-sulfoximine and its PEGylated form, and the glutathione S-transferase inhibitor is selected from glutathione analogs, ketoprofen, indomethacin, etacrynic acid, piroprofen, anti-GST antibody, and dominant negative mutants of GST, the antitumor agent according to any one of claims 1, 2, and 6.

8. The antitumor agent according to claim 7, wherein aldehyde dehydrogenase is highly expressed in the tumor cells.

9. The antitumor agent according to claim 7 or 8, wherein the tumor further contains tumor cells expressing CD44v.

10. A measuring method comprising the steps of simultaneously administering oxyphedrine or a pharmacologically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor to tumor cells in vitro, and measuring the growth rate or cell viability of the tumor cells, wherein the glutathione concentration reducing agent is selected from sulfasalazine, elastin, sorafenib, anti-xCT antibody, and L-buthionine-sulfoximine and its PEGylated form, and the glutathione S-transferase inhibitor is selected from glutathione analogs, ketoprofen, indomethacin, etacrynic acid, piroprofen, anti-GST antibody, and dominant negative mutants of GST.

11. A method for identifying a glutathione concentration reducing agent or a glutathione-S-transferase inhibitor having a combined effect with oxyphedrine or a pharmacologically acceptable salt thereof, comprising the steps of simultaneously administering oxyphedrine or a pharmacologically acceptable salt thereof and a plurality of glutathione concentration reducing agents or glutathione S-transferase inhibitors to tumor cells in vitro, and measuring the growth rate or cell viability of the tumor cells. The glutathione concentration reducing agent is selected from sulfasalazine, elastin, sorafenib, anti-xCT antibody, and L-buthionine-sulfoximine and its PEGylated form, The glutathione S-transferase inhibitor is selected from glutathione analogs, ketoprofen, indomethacin, etacrynic acid, piroprofen, anti-GST antibody, and dominant negative mutants of GST, by a specific method.

12. A specific method for tumor cells that exhibits a combined effect of oxyfedrine or a pharmaceutically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, A step of simultaneously administering a specific combination of oxyfedrine or a pharmaceutically acceptable salt thereof and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor to a plurality of tumor cells in vitro, A specific method comprising a step of measuring the growth rate or cell viability of the plurality of tumor cells, The glutathione concentration reducing agent is selected from sulfasalazine, elastin, sorafenib, anti-xCT antibody, and L-buthionine-sulfoximine and its PEGylated form, The glutathione S-transferase inhibitor is selected from glutathione analogs, ketoprofen, indomethacin, etacrynic acid, piroprofen, anti-GST antibody, and dominant negative mutants of GST, by a specific method.

13. The measuring method according to claim 10, wherein the tumor cells are resistant to a glutathione concentration reducing agent or a glutathione S-transferase inhibitor.

14. The tumor cells are resistant to a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, The glutathione concentration reducing agent is selected from sulfasalazine, elastin, sorafenib, anti-xCT antibody, and L-buthionine-sulfoximine and its PEGylated form, The specific method according to claim 11 or 12, wherein the glutathione S-transferase inhibitor is selected from glutathione analogs, ketoprofen, indomethacin, etacrynic acid, piroprofen, anti-GST antibody, and dominant negative mutants of GST.

15. An enhancer of antitumor action by co - administration of oxyphedrine or a pharmacologically acceptable salt thereof and a glutathione concentration - reducing agent or a glutathione S - transferase inhibitor, which contains an inhibitor of the enhancing action of Nrf2 on xCT expression, The glutathione concentration - reducing agent is selected from sulfasalazine, elastin, sorafenib, anti - xCT antibody, and L - butionine - sulfoximine and its PEGylated derivatives, The glutathione S - transferase inhibitor is selected from glutathione analogs, ketoprofen, indomethacin, etacrynic acid, piroprofen, anti - GST antibody, and dominant negative mutants of GST, The inhibitor of the enhancing action of Nrf2 on xCT expression is selected from antisense NA, miRNA, and siRNA against the Nrf2 gene, and ML385 and anti - Nrf2 antibody, and it is an enhancer of antitumor action.

16. The enhancer of antitumor action according to claim 15, wherein the antitumor action is an action against tumors overexpressing the Nrf2 gene.

17. The enhancer of antitumor action according to claim 15, wherein the glutathione concentration - reducing agent is sulfasalazine.

18. A companion diagnostic agent for predicting the antitumor effect by co - administration of oxyphedrine or a pharmacologically acceptable salt thereof and a glutathione concentration - reducing agent or a glutathione S - transferase inhibitor, The glutathione concentration - reducing agent is selected from sulfasalazine, elastin, sorafenib, anti - xCT antibody, and L - butionine - sulfoximine and its PEGylated derivatives, The glutathione S - transferase inhibitor is selected from glutathione analogs, ketoprofen, indomethacin, etacrynic acid, piroprofen, anti - GST antibody, and dominant negative mutants of GST, A companion diagnostic agent containing a reagent for detecting Nrf2 gene expression.

19. The companion diagnostic agent according to claim 18, wherein the detection reagent is an antibody, a probe for detecting gene expression, or a primer for gene amplification.

20. The companion diagnostic agent according to claim 18 or 19, wherein the glutathione concentration - reducing agent is sulfasalazine.

21. A companion diagnostic agent for predicting the antitumor effect by co - administration of oxyfedrine or a pharmacologically acceptable salt thereof and a glutathione concentration - reducing agent or a glutathione S - transferase inhibitor, wherein the glutathione concentration - reducing agent is selected from sulfasalazine, elastin, sorafenib, anti - xCT antibody, and L - buthionine - sulfoximine and its PEGylated form, wherein the glutathione S - transferase inhibitor is selected from glutathione analogs, ketoprofen, indomethacin, etacrynic acid, piroprostaglandin, anti - GST antibody, and dominant - negative mutants of GST, A companion diagnostic agent comprising a reagent for detecting mutations in the Keap1 gene or the Nrf2 gene.

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