Anti-tumor agent and compounding agent

Abstract

To provide novel anti-tumor agents and combination drugs.SOLUTION: Provided is an anti-tumor agent containing, as an active ingredient, a glutathione level reducer or a glutathione S-transferase inhibitor to be administered simultaneously with an effective amount of the following compound (II); an anti-tumor agent containing, as an active ingredient, the following compound (II) to be administered simultaneously with an effective amount of a glutathione level reducer or a glutathione S-transferase inhibitor; or an anti-tumor agent containing, as active ingredients, the following compound (II) and a glutathione level reducer or a glutathione S-transferase inhibitor. (In the formula, R5 is a C1-6 alkyl group; R6 and R8 are hydrogen or halogen; and R7 is a C1-6 alkyl group which may optionally have hydroxy or phenyl as a substituent).SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antitumor agents and combination agents. [Background technology]

[0002] In cancer treatment, the presence of cells that are resistant to treatments such as anticancer drugs and radiation can cause 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 that target cancer stem cells is urgently needed to cure cancer. However, analysis of the molecular mechanisms of stress resistance in cancer stem cells, which is necessary for the development of treatments that target cancer stem cells, has only just begun.

[0003] CD44, a marker of epithelial cancer stem cells, is known to be involved in their stress resistance (Cancer Cell. 2011 Mar 8;19(3):387-400). CD44 exists as a splice variant form (CD44v), which stably expresses the cystine transporter xCT on the cell membrane. xCT functions to import cystine into cells, which is then used to produce glutathione (GSH). Therefore, cells with high CD44v expression have increased GSH levels. GSH has a strong antioxidant effect and plays a role in reducing cellular stress. Therefore, cancer stem cells with high CD44v expression are thought to be resistant to treatment.

[0004] Meanwhile, sulfasalazine (also known as salazosulfapyridine, salazopyrin, and salicylazosulfapyridine) is a drug used to treat ulcerative colitis and rheumatoid arthritis. Sulfasalazine is an acidic azo compound consisting of sulfapyridine and 5-aminosalicylic acid (5-ASA). When administered orally, it is broken down into sulfapyridine and 5-aminosalicylic acid (5-ASA) by intestinal bacteria in the intestine. 5-ASA is considered to be the main active ingredient in these diseases.

[0005] Recently, it has been revealed that the unchanged sulfasalazine before degradation has an xCT inhibitory effect and is effective as an antitumor agent (Leukemia vol.15, pp.1633-1640, 2001). In other words, when sulfasalazine is added to cancer cells, the uptake of cystine into the cells by xCT is suppressed, reducing glutathione production, which in turn reduces the resistance of cancer cells to oxidative stress and increases their sensitivity to antitumor agents.

[0006] Sulfasalazine, which has an xCT inhibitory effect, is known to effectively suppress the proliferation of cancer stem cells that highly express CD44v (JP 2012-144498 A). Summary of the Invention [Problem to be solved by the invention]

[0007] An object of the present invention is to provide a novel antitumor agent and a combination drug. [Means for solving the problem]

[0008] The present inventors have found that sulfasalazine alone has an antitumor effect against tumors composed mostly of undifferentiated tumor cells, but that it does not have the effect of reducing the overall tumor volume against differentiated tumors containing tumor cells exhibiting differentiated phenotypes, although it reduces cancer stem cells highly expressing CD44v. Therefore, the present inventors have made intensive efforts to develop an antitumor agent for differentiated tumors by developing a drug that has an antitumor effect against tumor cells on which sulfasalazine has no antitumor effect. As a result, they have found that the combined use of an aldehyde dehydrogenase inhibitor or oxyfedrine with sulfasalazine or L-buthionine sulfoximine, respectively, has a significant antitumor effect against tumor cells to which sulfasalazine or L-buthionine sulfoximine alone has only a weak effect, leading to the completion of the present invention.

[0009] One embodiment of the present invention is an antitumor agent comprising, as an active ingredient, a glutathione concentration-lowering 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 pharmacologically acceptable salt thereof:

[0010] JPEG2025114717000001.jpg44170 (in the formula, R 5 is a C1-6 linear or branched alkyl group, and R 6 is hydrogen or halogen, and R 7 is a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, and R 8 is hydrogen or 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 pharmacologically acceptable salt thereof, which is administered simultaneously with an effective amount of a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor:

[0011] JPEG2025114717000002.jpg44170 (in the formula, R 5 is a C1-6 linear or branched alkyl group, and R2 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 ring with the N to which they are attached as a heteroatom, and R 4 is hydrogen or halogen.) In any of the above antitumor agents, the glutathione concentration-lowering agent may be a drug that inhibits the activity of xCT, thioredoxin-1 (TRX-1), glutamate-cysteine ligase (GCL) (EC 6.3.2.2) (also known as γ-glutamylcysteine synthetase), or glutathione synthetase (EC 6.3.2.3). The drug may be an xCT or GCL inhibitor, or may be sulfasalazine or L-buthionine sulfoximine. The compound represented by formula (II) may be oxyfedrine.

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

[0013] JPEG2025114717000003.jpg44170 (in the formula, R 5 is a C1-6 linear or branched alkyl group, and R 6 is hydrogen or halogen, and R 7 is a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, and R 8 is hydrogen or halogen.) In the above-mentioned combination drug, the glutathione concentration-lowering agent may be a drug that inhibits the activity of xCT, thioredoxin-1 (TRX-1), glutamate-cysteine ligase (GCL) (EC 6.3.2.2) (also known as γ-glutamylcysteine synthetase), or glutathione synthetase (EC 6.3.2.3). The drug may be an xCT or GCL inhibitor, sulfasalazine or a derivative thereof, or L-buthionine sulfoximine. The compound represented by formula (II) may be oxyfedrine.

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

[0015] Any of the above antitumor agents may be used for tumors containing tumor cells resistant to glutathione concentration-lowering agents or glutathione S-transferase inhibitors. The tumor cells may have high expression of aldehyde dehydrogenase. The tumor may further contain tumor cells expressing CD44v.

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

[0017] JPEG2025114717000004.jpg42170 (wherein X is hydrogen, halogen, -NH2, or -CN, Y is a C1-6 linear or branched alkyl group, and Z 1 and Z 2 are each hydrogen or halogen, and a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, or Z 1 and Z 2together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom. In this measurement method, the tumor cells may be resistant to a glutathione concentration-lowering 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-lowering agent or a glutathione S-transferase inhibitor, the method comprising the steps of simultaneously administering a specific glutathione concentration-lowering agent or glutathione S-transferase inhibitor and each of multiple aldehyde dehydrogenase inhibitors, or compound (III) or a pharmacologically acceptable salt thereof, to tumor cells in vitro, and measuring the proliferation rate or cell viability of the tumor cells.

[0019] JPEG2025114717000005.jpg42170 (wherein X is hydrogen, halogen, -NH2, or -CN, Y is a C1-6 linear or branched alkyl group, and Z 1 and Z 2 are each hydrogen or halogen, and a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, or Z 1 and Z 2 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom. A further embodiment of the present invention is a method for identifying a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor having a combined effect with the antitumor agent compound (III) or a pharmaceutically acceptable salt thereof, the method comprising the steps of simultaneously administering compound (III) and a plurality of glutathione concentration-lowering agents or glutathione S-transferase inhibitors to tumor cells in vitro, and measuring the proliferation rate or cell viability of the tumor cells.

[0020] JPEG2025114717000006.jpg42170 (wherein X is hydrogen, halogen, -NH2, or -CN, Y is a C1-6 linear or branched alkyl group, and Z 1 and Z 2 are each hydrogen or halogen, and a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, or Z 1 and Z 2 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom. The compound (III) may be the compound (II).

[0021] JPEG2025114717000007.jpg44170 (in the formula, R 5 is a C1-6 linear or branched alkyl group, and 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 ring with the 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 method for identifying tumor cells that exhibit the combined effect of the antitumor agent compound (III) or a pharmacologically acceptable salt thereof with a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, the method comprising the steps of simultaneously administering a specific combination of compound (III) or a pharmacologically acceptable salt thereof with a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor to a plurality of tumor cells in vitro, and measuring the proliferation rate or cell viability of the plurality of tumor cells.

[0022] JPEG2025114717000008.jpg42170 (wherein X is hydrogen, halogen, -NH2, or -CN, Y is a C1-6 linear or branched alkyl group, and Z 1and Z 2 are each hydrogen or halogen, and a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, or Z 1 and Z 2 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom. The compound (III) may be the compound (II).

[0023] JPEG2025114717000009.jpg44170 (in the formula, R 5 is a C1-6 linear or branched alkyl group, and 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 ring with the N to which they are attached as a heteroatom, and R 4 is hydrogen or halogen.) In any particular method above, the tumor cells may be resistant to glutathione concentration-lowering agents or glutathione S-transferase inhibitors. A further embodiment of the present invention is an antitumor agent for use in radiation therapy, which contains an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof as an active ingredient. JPEG2025114717000010.jpg44170 (in the formula, R 5 is a C1-6 linear or branched alkyl group, and 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 ring with the N to which they are attached as a heteroatom, and R 4is hydrogen or halogen.) The compound represented by formula (II) may be oxyfedrine. It may also be an antitumor agent for tumors containing tumor cells resistant to glutathione concentration-lowering agents or glutathione S-transferase inhibitors. 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 antitumor effect, comprising the steps of irradiating tumor cells in vitro in the presence of an aldehyde dehydrogenase inhibitor or compound (III) or a pharmaceutically acceptable salt thereof, and measuring the proliferation rate or cell survival rate of the tumor cells. JPEG2025114717000011.jpg42170 (wherein X is hydrogen, halogen, -NH2, or -CN, Y is a C1-6 linear or branched alkyl group, and Z 1 and Z 2 are each hydrogen or halogen, and a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, or Z 1 and Z 2 are taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom.) The tumor cells may be resistant to glutathione concentration-lowering agents or glutathione S-transferase inhibitors. A further embodiment of the present invention is a method for identifying an agent that has a synergistic effect with radiation on tumor cells in vitro, the method comprising the steps of irradiating tumor cells in vitro in the presence of an aldehyde dehydrogenase inhibitor or each of a plurality of Compound (III) or a pharmacologically acceptable salt thereof, and measuring the proliferation rate or cell viability of the tumor cells. JPEG2025114717000012.jpg42170 (wherein X is hydrogen, halogen, -NH2, or -CN, Y is a C1-6 linear or branched alkyl group, and Z 1 and Z 2are each hydrogen or halogen, and a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, or Z 1 and Z 2 are taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group in which the N to which they are attached is a heteroatom.) Compound (III) may be compound (II). JPEG2025114717000013.jpg44170 (in the formula, R 5 is a C1-6 linear or branched alkyl group, and 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 ring with the N to which they are attached as a heteroatom, and R 4 is hydrogen or halogen.) The tumor cells may be resistant to glutathione concentration-lowering agents or glutathione S-transferase inhibitors. A further embodiment of the present invention is an agent for enhancing antitumor activity by co-administering an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof with a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, the agent comprising an inhibitor of the xCT expression-enhancing activity of Nrf2: JPEG2025114717000014.jpg44170 (in the formula, R 5 is a C1-6 linear or branched alkyl group, and R 6 is hydrogen or halogen, and R 7 is a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, and R 8is hydrogen or halogen. The inhibitor may be an expression inhibitor of the Nrf2 gene or an Nrf2 inhibitor. The expression inhibitor of the Nrf2 gene may be an antisense NA, miNA, or siNA against the Nrf2 gene. The Nrf2 inhibitor may be ML385 or an anti-Nrf2 antibody. The antitumor effect may be an effect on tumors overexpressing the Nrf2 gene. The glutathione concentration-lowering agent may be sulfasalazine. The compound represented by formula (II) may be oxyfedrine. A further embodiment of the present invention is a companion diagnostic agent for predicting the antitumor effect of co-administration of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof with a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, the companion diagnostic agent comprising a reagent for detecting Nrf2 gene expression. JPEG2025114717000015.jpg44170 (in the formula, R 5 is a C1-6 linear or branched alkyl group, and R 6 is hydrogen or halogen, and R 7 is a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, and R 8 is hydrogen or halogen. The detection reagent may be an antibody, a probe for detecting gene expression, or a primer for gene amplification. The glutathione concentration-lowering agent may be sulfasalazine. The compound represented by formula (II) may be oxyfedrine. A further embodiment of the present invention is a companion diagnostic agent for predicting the antitumor effect of co-administration of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof with a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, the companion diagnostic agent comprising a reagent for detecting mutations in the Keap1 gene or the Nrf2 gene. JPEG2025114717000016.jpg44170 (in the formula, R 5is a C1-6 linear or branched alkyl group, and R 6 is hydrogen or halogen, and R 7 is a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, and R 8 is hydrogen or halogen.) ==Cross-reference to related literature== 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 these basic applications are incorporated herein by reference. [Brief explanation of the drawings]

[0024] [Figure 1] 1 is a graph showing the effect of combined use of sulfasalazine and dyclonine in one example of the present invention. [Figure 2] 1 is a graph showing changes in dyclonine sensitivity due to xCT knockdown in one example of the present invention. [Figure 3] FIG. 1 is a graph showing the combined effects of sulfasalazine, erastin, or BSO with dyclonine in various cancer cell lines in one example of the present invention. [Figure 4] 1 is a graph showing the in vivo effect of combined use of sulfasalazine and dyclonine in one example of the present invention. [Figure 5] FIG. 1 is a graph showing experimental results showing the inhibitory effect of dyclonine on ALDH activity in one example of the present invention. [Figure 6] FIG. 1 is a graph showing the accumulation effect of HNE (4-HNE; 4-hydroxy-2-nonenal) when sulfasalazine and dyclonine are used in combination in one example of the present invention. [Figure 7] 1 is a graph showing the effect of combined use of sulfasalazine or BSO with a dyclonine analog (having a dyclonine skeleton) in one example of the present invention. [Figure 8]1 is a graph showing the effect of combined use of BSO and a dyclonine analog (not having a dyclonine skeleton) in one example of the present invention. [Figure 9] 1 is a graph showing the combined effects of sulfasalazine, erastin, or BSO with dyclonine on OSC19 cells or sulfasalazine-resistant OSC19 cells in one example of the present invention. [Figure 10] 1 is a graph showing the expression of the ALDH gene family in HSC4 cells, OSC19 cells, or sulfasalazine-resistant OSC19 cells in one example of the present invention. [Figure 11] FIG. 1 is a graph showing experimental results showing the combined effect of oxyfedrine with sulfasalazine (SSZ) or L-buthionine sulfoximine (BSO) in one example of the present invention. [Figure 12] Figure 1 shows the metabolic pathway of HNE. Abbreviations: HNA, 4-hydroxy-2-nonenoic acid; GSH, glutathione; ALDH, aldehyde dehydrogenase; GST, glutathione S-transferase. [Figure 13] FIG. 1 is a graph showing a decrease in GSH concentration in tumor cells by sulfasalazine or BSO in one example of the present invention. [Figure 14] FIG. 1 shows the effect of combined use of sulfasalazine or BSO with oxyfedrine on intracellular HNE accumulation in one example of the present invention. [Figure 15] 1 is a graph showing the effect of combined use of sulfasalazine and oxyfedrine on reducing tumor cell viability in vivo in one example of the present invention. [Figure 16] 1 is a graph showing the combined effect of sulfasalazine and oxyfedrine on intracellular HNE accumulation in vivo in one example of the present invention. [Figure 17] FIG. 1 is a graph showing experimental results showing the combined effect of radiation exposure and oxyfedrine administration on reducing tumor cell viability in one example of the present invention. [Figure 18]FIG. 1 is a graph showing experimental results showing the combined effect of radiation exposure and oxyfedrine administration on intracellular HNE accumulation in one example of the present invention. [Figure 19] FIG. 1 shows experimental results showing the expression of Nrf2 and xCT in SSZ-resistant tumor cells in one example of the present invention. [Figure 20] FIG. 1 is a graph showing the experimental results showing the cell viability of A549 cells grown in a medium containing siRNA against the Nrf2 gene or ML385, an Nrf2 inhibitor, OXY, and SSZ or BSO, in one example of the present invention. [Figure 21] FIG. 1 is a graph showing experimental results showing the combined effect of radiation irradiation and oxyfedrine (OXY) administration on the suppression of tumor cell weight increase in vivo in one example of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0025] Hereinafter, embodiments of the present invention will be described in detail with reference to examples. The objects, features, advantages, and concepts of the present invention will be apparent to those skilled in the art from the description in this specification, and those skilled in the art will be able to 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 illustrative or explanatory purposes, and are not intended to limit the present invention. It will be apparent to those skilled in the art that various changes and modifications can be made based on the description in this specification within the intent and scope of the present invention disclosed herein.

[0026] Unless otherwise specified in the embodiments and examples, the methods described in standard protocol collections or modified or altered methods are used. Furthermore, when commercially available reagent kits or measuring devices are used, the protocols attached to them are used unless otherwise specified.

[0027] ==Antineoplastic Agents== One embodiment of the present invention is an antitumor agent containing, as an active ingredient, a glutathione concentration-lowering agent, which is administered simultaneously with an effective amount of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof. Here, the effective amount of the aldehyde dehydrogenase inhibitor is an amount that has a combined effect with the glutathione concentration-lowering agent in terms of 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 pharmacologically acceptable salt thereof, which is administered simultaneously with an effective amount of a glutathione concentration-lowering agent. Here, the effective amount of the glutathione concentration-lowering agent is an amount of the glutathione concentration-lowering agent that has a combined effect with the aldehyde dehydrogenase inhibitor in terms of antitumor activity.

[0029] Without being bound by the following theory, as shown in Figure 12, there are multiple pathways for degrading HNE within cells, and it is believed that simultaneous inhibition of both the GST-mediated pathway and the ALDH-mediated pathway leads to intracellular accumulation of HNE. Furthermore, because HNE is cytotoxic, it is believed that tumor cells are unable to proliferate. Therefore, simultaneous administration of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof with a glutathione concentration-lowering agent exerts a synergistic antitumor effect.

[0030] Another embodiment of the present invention is an antitumor agent for use in radiation therapy, containing as an active ingredient an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof. Radiation therapy is a therapeutic method used to treat tumors, and the dose and method of irradiation can be easily determined by the therapist based on common general knowledge, the condition of the tumor, the patient, and other factors. It is known that radiation exposure reduces intracellular GSH levels, and the combined effects of both can be achieved by irradiating a tumor patient in the presence of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof. Therefore, in this case, the tumor patient may be administered an antitumor agent and radiation exposure may be continued while the concentration is such that a synergistic effect with radiation exposure is observed. Alternatively, the tumor patient may be irradiated and the antitumor agent may be administered while GSH levels are decreasing.

[0031] Aldehyde dehydrogenase inhibitors are drugs that inhibit the enzymatic activity of aldehyde dehydrogenase 2 (ALDH) (EC 1.2.1.10). The type and isotype of ALDH to be inhibited are not particularly limited, and may be any of ALDH1 to 5 and their isotypes. Aldehyde dehydrogenase inhibitors used as antitumor agents are not particularly limited, and examples thereof include chlorpropamide, tolbutamide, diethylaminobenzaldehyde, disulfiram (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 analogs thereof, or pharmacologically acceptable salts thereof. In particular, dyclonine and dyclonine analogs (I) shown below are preferred, and the compounds shown in Figure 7 (BAS00363846, STL327701, PHAR033081, PHAR298639, and Aldi-2) are more preferred.

[0032] JPEG2025114717000017.jpg43170 (in the formula, R 1 is a C1-6 linear or branched alkyl group, and 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 ring with the N to which they are attached as a heteroatom, and R 4is hydrogen or halogen. 1 is preferably a C4-5 linear or branched alkyl group, and R 2 and R 3 is a C2 alkyl group, or R 2 and R 3 are preferably taken together to form a 6-membered azacycloalkyl group with N as the hetero atom to which they are attached. 1 is a C4 linear alkyl group, and R 2 and R 3 The compound in which the groups taken together form a six-membered azacycloalkyl group with the N to which they are attached as a heteroatom is dyclonine. The halogen is preferably F, Cl, I, Br, or I. Compound (II) is oxyfedrine or its analogues and has the following structural formula:

[0033] JPEG2025114717000018.jpg44170 (in the formula, R 5 is a C1-6 linear or branched alkyl group, and R 6 is hydrogen or halogen, and R 7 is a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, and R 8 is hydrogen or halogen.) The compound (II) used is preferably oxyfedrine having the following structural formula (IV), and the salt thereof is preferably oxyfedrine hydrochloride.

[0034] JPEG2025114717000019.jpg49170 Note that the pharmacologically acceptable salt is not limited as long as it forms a salt with these compounds, and specific 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, with hydrochloride, oxalate, maleate, and methanesulfonate being preferred. Furthermore, it goes without saying that these compounds or their pharmacologically acceptable salts include not only anhydrides but also hydrates and crystalline polymorphs.

[0035] Glutathione concentration-lowering agents are drugs that reduce intracellular glutathione concentrations. While glutathione concentration-lowering agents used in these antitumor agents are not limited, agents that inhibit the pathway by which glutathione is produced from cystine taken up into cells by xCT are preferred. Agents that inhibit the activity of xCT, thioredoxin-1 (TRX-1), glutamate-cysteine ligase (GCL) (EC 6.3.2.2) (also known as γ-glutamylcysteine synthetase), or glutathione synthetase (EC 6.3.2.3) are more preferred, with xCT inhibitors or GCL inhibitors being more preferred. While xCT inhibitors are not particularly limited, sulfasalazine, erastin, sorafenib, or derivatives thereof, or anti-xCT antibodies are preferred. While GCL inhibitors are also not particularly limited, L-buthionine sulfoximine or a derivative thereof is preferred. Derivatives are not limited to glutathione concentration-lowering agents, but examples include PEGylated derivatives.

[0036] Glutathione S-transferase inhibitors are drugs that inhibit the enzymatic activity of glutathione S-transferase (EC 2.5.1.18), particularly the activity of converting HNE (4-HNE; 4-hydroxy-2-nonenal) to HNE-GSH. Glutathione S-transferase inhibitors are not particularly limited, but examples include glutathione analogs (e.g., WO95 / 08563, WO96 / 40205, WO99 / 54346, etc.), ketoprofen, indomethacin, ethacrynic acid, piroprost, anti-GST antibodies, and dominant-negative mutants of GST.

[0037] As used herein, "simultaneous administration" of two or more drugs refers not only to administration at the same time, but also to administration of each drug separately at different times, as long as the effects of one drug are still being administered while the other drug is still active. When administering two or more drugs simultaneously, two or more drugs may be administered simultaneously, or two or more drugs may be administered in a single dosage form as a combined drug. Note that "coadministration" and "coadminister" are also intended to have the same meaning as "simultaneous administration" and "administer simultaneously," respectively.

[0038] The subject of administration of the antitumor agent is not particularly limited as long as it is a vertebrate, but is preferably a human cancer patient. The tumor to be treated is not particularly limited, but tumors containing tumor cells resistant to glutathione concentration-lowering agents or glutathione S-transferase inhibitors are preferred. These tumor cells may have high expression of aldehyde dehydrogenase. The glutathione concentration-lowering agent or glutathione S-transferase inhibitor is preferably an xCT inhibitor, more preferably sulfasalazine. Resistant tumor cells are tumor cells that survive when administered to a patient in vivo at a normal therapeutic concentration for a normal number of days of treatment, 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 types of cell lines. For example, sulfasalazine-resistant tumor cells are tumor cells that survive when administered to a patient in vivo at a dose of 50% or less of AUC 0-24Tumor cells that survive when administered at 50-300 μg h / mL for approximately 2 weeks, and in vitro, the survival rate at 200 μM is 90% or more. In addition, L-buthionine-sulfoximine-resistant cells can be measured in vivo in patients with AUC 0-24 Tumor cells that survive administration of 10 to 100 μg h / mL for approximately two weeks and have a survival rate of 90% or more in vitro at 100 μM can be used. Sulfasalazine-resistant tumor cells and L-buthionine sulfoximine-resistant cells preferably also have low levels of CD44v expression or are negative. Tumor cells overexpressing aldehyde dehydrogenase refer to cells expressing any of the genes ALDH1A1, ALDH2, ALDH1B1, and ALDH3A1 at levels three-fold or more, preferably ten-fold higher, than OSC19 cells. The tumor to be treated may contain tumor cells expressing CD44v. This is because sulfasalazine and L-buthionine sulfoximine have effective antitumor effects on tumor cells expressing CD44v. Tumor cells expressing CD44v may be any cells in which CD44v expression can be detected, but cells with high expression are preferred. In this case, high expression may be the same as or higher than the average level of ovarian tumor cells, but is preferably at least two times higher, more preferably at least four times higher, and even more preferably at least ten times higher.

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

[0040] The antitumor agent may be formulated into tablets, powders, granules, dispersing agents, capsules, liquids, emulsions, suspensions, etc. by a conventional method, using pharmaceutically acceptable additives known to those skilled in the art, such as excipients and carriers.

[0041] The antitumor agent may be administered within the effective dose range in a manner appropriate for the subject. The effective dose can be ultimately determined appropriately by the judgment of a physician or veterinarian, taking into consideration the type of dosage form, administration method, the age and weight of the subject, and the condition of the subject. For example, the daily dose of the compound is preferably 0.1 mg / kg or more, more preferably 1 mg / kg or more, and even more preferably 10 mg / kg or more, and 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, and may be, for example, oral administration, parenteral administration by intraperitoneal or intravenous injection or infusion, or direct administration into the cancer by injection or the like.

[0042] ==Antitumor effect enhancer of antitumor agents== One embodiment of the present invention is an antitumor effect enhancer obtained by co-administering an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof with a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, and the enhancer contains an inhibitor of the xCT expression-enhancing function of Nrf2. It can also be said to be an antitumor effect enhancer of the antitumor agent described above. This antitumor effect enhancer of an antitumor agent is administered simultaneously with the antitumor 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 thought that suppressing the expression level of Nrf2 suppresses the expression of xCT and ALDH, thereby enhancing the effect of antitumor agents.

[0044] Examples of inhibitors of the xCT expression-enhancing effect of Nrf2 include substances that suppress the expression of the Nrf2 gene and inhibitors of the xCT expression-enhancing function of Nrf2. That is, to suppress the xCT expression-enhancing effect of Nrf2, the expression of the Nrf2 gene may be suppressed in tumor cells, or the xCT expression-enhancing function of Nrf2 as a protein may be inhibited.

[0045] Examples of substances that suppress the expression of the Nrf2 gene include antisense NAs, miNAs, and siNAs against the Nrf2 gene. Each of these may consist of RNA, DNA, or a chimeric molecule of RNA and DNA. Nucleic acids (NAs) may also contain various modifications. Their sequences can be easily designed by those skilled in the art using their common technical knowledge. Examples of Nrf2 inhibitors include low-molecular-weight compounds such as ML385 and anti-Nrf2 antibodies.

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

[0047] ==Methods for measuring tumor cell proliferation rate or cell viability== One embodiment of the present invention is a measurement method comprising the steps of simultaneously administering an aldehyde dehydrogenase inhibitor, or compound (III) or a pharmacologically acceptable salt thereof, and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor to tumor cells in vitro, and measuring the proliferation rate or cell viability of the drug-administered tumor cells. Another embodiment of the present invention is a measurement method comprising the steps of irradiating tumor cells in vitro in the presence of an aldehyde dehydrogenase inhibitor or compound (III) or a pharmaceutically acceptable salt thereof, and measuring the proliferation rate or cell viability of the tumor cells. The aldehyde dehydrogenase inhibitors, glutathione concentration-lowering agents, and glutathione S-transferase inhibitors in this section are the same as those described in detail in the "Antineoplastic Agents" section. Compound (III) has the following structural formula, but is preferably compound (II).

[0048] JPEG2025114717000020.jpg42170 (wherein X is hydrogen, halogen, -NH2, or -CN, Y is a C1-6 linear or branched alkyl group, and Z 1 and Z 2 are each hydrogen or halogen, and a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl, or Z 1 and Z 2 together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom. An aldehyde dehydrogenase inhibitor, or compound (III) or a pharmacologically acceptable salt thereof, and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor have a combined antitumor activity effect, and therefore, this measurement method can be used to find drug combinations that have a high combined effect, or to find tumor cells that are particularly effective against a certain drug combination.

[0049] Specifically, a specific glutathione concentration-lowering agent or glutathione S-transferase inhibitor and multiple aldehyde dehydrogenase inhibitors or compound (III) or a pharmacologically acceptable salt thereof are simultaneously administered to tumor cells in vitro, and the proliferation rate or cell viability of the drug-administered tumor cells is measured, thereby enabling identification of an aldehyde dehydrogenase inhibitor or compound (III) or a pharmacologically acceptable salt thereof that has a combined effect with a specific glutathione concentration-lowering agent or glutathione S-transferase inhibitor. Furthermore, a specific aldehyde dehydrogenase inhibitor or compound (III) or a pharmacologically acceptable salt thereof and multiple glutathione concentration-lowering agents or glutathione S-transferase inhibitors that have a combined effect with a specific aldehyde dehydrogenase inhibitor or compound (III) or a pharmacologically acceptable salt thereof can be identified by simultaneously administering a specific aldehyde dehydrogenase inhibitor or compound (III) or a pharmacologically acceptable salt thereof to tumor cells in vitro, and measuring the proliferation rate or cell viability of the drug-administered tumor cells. Alternatively, a drug having a synergistic effect with radiation can be identified by irradiating tumor cells in vitro in the presence of an aldehyde dehydrogenase inhibitor or each of multiple compounds (III) or pharmacologically acceptable salts thereof, and measuring the proliferation rate or cell survival rate of the tumor cells.

[0050] Furthermore, by simultaneously administering a specific combination of an aldehyde dehydrogenase inhibitor, or compound (III) or a pharmacologically acceptable salt thereof, and a glutathione concentration-lowering agent or glutathione S-transferase inhibitor to multiple tumor cells in vitro and measuring the proliferation rate or cell viability of the multiple drug-treated tumor cells, it is possible to identify tumor cells that exhibit the combined effect of an aldehyde dehydrogenase inhibitor, or compound (III) or a pharmacologically acceptable salt thereof, and a glutathione concentration-lowering agent or glutathione S-transferase inhibitor. Compound (III) used in these methods is preferably an antitumor agent having antitumor activity.

[0051] ==Companion Diagnostic Agents== One embodiment of the present invention is a companion diagnostic agent for predicting the antitumor effect of the above-mentioned antitumor agent, which comprises a reagent for detecting Nrf2 gene expression. In recent years, it has become known that Nrf2 expression is a factor that contributes to tumor malignancy. As shown in the Examples, tumor cells with high Nrf2 expression levels are relatively less effective against antitumor agents. While not being bound by this theory, there is a positive correlation between the Nrf2 expression level and the expression levels of xCT and ALDH. Since the above-mentioned antitumor agents simultaneously suppress the expression of xCT and ALDH, it is thought that a high Nrf2 expression level makes it difficult for the antitumor agents to exert their effects. 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. Nrf2 gene expression can be detected at any stage leading up to the final product, Nrf2 protein, for example, by detecting mRNA or protein. Reagents for detecting Nrf2 gene expression are not particularly limited and can be easily selected based on common technical knowledge, but may include, for example, antibodies, gene expression detection probes, or gene amplification primers. Those skilled in the art can easily prepare anti-Nrf2 antibodies and design gene expression detection probes and gene amplification primers based on common technical knowledge. Furthermore, because constitutive expression of the Nrf2 protein may involve mutations in the Keap1 and Nrf2 genes, the companion diagnostic agent is also a reagent for detecting known mutations in the Keap1 or Nrf2 gene. Detection of mutations in the Keap1 or Nrf2 gene can be performed by known techniques, and may include, for example, gene amplification primers for amplifying the Keap1 or Nrf2 gene. [Example]

[0052] (Experimental Example 1) Effect of combined use of sulfasalazine and dyclonine (Purpose) This experiment demonstrates that the combined use of sulfasalazine, which has an xCT inhibitory effect, and dyclonine has an effect on the reduction in the viability of sulfasalazine-resistant cells.

[0053] (Method) Oral squamous cell carcinoma cell line HSC-4, a sulfasalazine-resistant cell line, was seeded at 2000 cells / well in a 96-well plate and culture was initiated. DMEM medium was used. After 24 hours, the medium was replaced with medium containing 50 μM dyclonine or an equivalent amount of DMSO and 0 μM (no addition), 50 μM, 100 μM, 200 μM, or 400 μM sulfasalazine, and culture was continued for 48 hours. Cell viability was then measured using Celltiter-Glo (Promega). The cell viability for each case was calculated, with the cell viability for the control (DMSO-added, no sulfasalazine) set at 100%. Figure 1 shows a graph showing the viability for each concentration of sulfasalazine.

[0054] (Results) HSC4 is a sulfasalazine-resistant cell line, and sulfasalazine alone has little effect on cell viability. Furthermore, dyclonine alone (with dyclonine, but without sulfasalazine) showed a viability of 80%. However, when both dyclonine and sulfasalazine were added, the viability fell to less than 10% at sulfasalazine concentrations above 100 μM.

[0055] Thus, the combined use of sulfasalazine and dyclonine is effective in reducing the viability of sulfasalazine-resistant cells.

[0056] (Experimental Example 2) Changes in dyclonine sensitivity due to xCT knockdown (Purpose) This experimental example demonstrates that knocking down xCT instead of sulfasalazine, which has an xCT inhibitory effect, can produce a similar combined effect with dyclonine, thereby demonstrating that the combined effect of sulfasalazine and dyclonine is mediated by the xCT inhibitory effect of sulfasalazine.

[0057] (Method) HSC-4 cells, a sulfasalazine-resistant oral squamous cell carcinoma cell line, were seeded at 3,000 cells / well in a 96-well plate and lipofected with a non-silencing control (scrambled [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)) using Lipofectamine RNAiMAX (ThermoFisher Scientific) and culture was initiated in DMEM medium. After 24 hours, the medium was replaced with 50 μM dyclonine (solvent: DMSO) or an equivalent amount of DMSO, and the cells were cultured for 48 hours. Cell viability was then measured using Celltiter-Glo (Promega). The cell viability of the control (non-silencing control, DMSO-treated) was set at 100%, and the cell viability of each cell was calculated. The results are shown in Figure 2.

[0058] (Results) HSC-4 cells had a cell viability of approximately 60% with 50 μM dyclonine alone, whereas when xCT was knocked down, the cell viability was only approximately 10-20% in the presence of 50 μM dyclonine.

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

[0060] (Experimental Example 3) Effect of combined use of sulfasalazine, erastin, or BSO with dyclonine on various cancer cell lines (Objective) This experimental example demonstrates the combined effects of sulfasalazine, erastin, a specific inhibitor of xCT, or BSO, a glutathione synthesis inhibitor, with dyclonine in various tumor cell lines, and also demonstrates that the inhibition of xCT is mediated by inhibition of glutathione synthesis.

[0061] (Method) The cell lines shown in Figure 3 were seeded at 3,000 cells / well in a 96-well plate and culture was initiated. DMEM was used as the culture medium. After 24 hours, the medium was replaced with one containing 50 μM dyclonine or an equivalent amount of DMSO, 0 μM (no addition) or 400 μM sulfasalazine, 0 μM (no addition) or 5 μM elastin, and 0 μM (no addition) or 100 μM BSO, and culture was continued for 48 hours. Cell viability was then measured using Celltiter-Glo (Promega). The cell viability for each case was calculated, with the number of viable cells in the control (DMSO-added, no dyclonine) set as 100%. Figure 3 shows the cell viability for each case.

[0062] (Results) Although the effect varied depending on the cell type, similar combined effects were observed with sulfasalazine, erastin, or BSO and dyclonine.

[0063] Thus, the antitumor effects of sulfasalazine and erastin on xCT inhibition are due to the inhibition of glutathione synthesis, and glutathione concentration-lowering agents or glutathione S-transferase inhibitors can be used instead of sulfasalazine and erastin.

[0064] (Experimental Example 4) In vivo effects of combined use of sulfasalazine and dyclonine (Objective) This experimental example demonstrates that the combined effects of sulfasalazine and dyclonine can be observed in vivo.

[0065] (Method) 1 × 10 cells of HSC-2, a sulfasalazine-resistant oral squamous cell carcinoma cell line, were used. 6The tumors were implanted subcutaneously into nude mice, and starting on day 4 after implantation, saline, sulfasalazine alone, dyclonine alone, or both sulfasalazine and dyclonine were administered intraperitoneally once daily at doses of 400 mg / kg sulfasalazine and 5 mg / kg dyclonine, until day 22. The tumor's short and long diameters were measured every 3 to 4 days, and tumor volume was calculated using the following formula. The results are graphed in Figure 4.

[0066] Tumor volume = (longer diameter x (short diameter) 2 ) / 2 Statistical analysis of tumor volume was performed on day 22 using t-test.

[0067] (Results) As shown in Figure 4, administration of each drug alone reduced tumor volume by approximately 35%, but administration of both drugs reduced the volume by approximately 70%.

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

[0069] (Experimental Example 5) Inhibition of ALDH by Dyclonine (Objective) This experimental example demonstrates that dyclonine has ALDH inhibitory activity.

[0070] (Method) Oral squamous cell carcinoma cell line HSC-4 cells were cultured in a 10 cm cell culture dish at 8 × 10 5Cells were seeded at 1000 cells / dish and cultured. DMEM was used. After 24 hours, the medium was replaced with one containing 50 μM dyclonine (solvent: DMSO) and cultured for another 24 hours. Cells were then harvested and stained for ALDH activity in the presence of N,N-diethylaminobenzaldehyde (DEAB) using the ALDEFLUOR kit (STEMCELL Technologies) and analyzed by FACS (Dyclonine in the figure). Controls include a sample without DEAB and not stained with the ALDEFLUOR kit (Unstained in the figure), and a sample with the medium replaced with an equal amount of DMSO without dyclonine and stained with the ALDEFLUOR kit (Non-treatment in the figure). For the measurement of positive cells, a gate was created so that the percentage of positive cells was approximately 0% for the DMSO-treated sample stained with the ALDEFLUOR kit in the presence of DEAB (DEAB in the figure), and the positive rate for each case was calculated.

[0071] (Results) As shown in Figure 5, in DMSO-treated cells, the cell population with high ALDH activity was approximately 25%, whereas in dyclonine-treated cells and cells treated with DEAB, a known ALDH inhibitor, the cell population with high ALDH activity was suppressed to approximately 1%.

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

[0073] (Experimental Example 6) HNE accumulation caused by combined use of sulfasalazine and dyclonine (Objective) This experimental example demonstrates 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) As in Experimental Example 1, HSC-4 cells were cultured in medium containing 50 μM dyclonine or an equivalent amount of DMSO and 0 μM (no additive) or 400 μM sulfasalazine. The treated cells were fixed in 4% PFA-PBS. Furthermore, the cell membrane was permeabilized with 0.2% TritonX100-PBS, followed by blocking with 3% BSA-PBS. Subsequently, fluorescent staining was performed using anti-HNE antibody as the primary antibody and Alexafluor 488-labeled anti-mouse IgG antibody as the secondary antibody. As a positive control, cells incubated with 50 μM HNE for 30 minutes were similarly stained with antibodies. Images observed under a fluorescent microscope are shown in Figure 6.

[0075] (Results) When dyclonine or sulfasalazine was administered alone, an increase in intracellular HNE concentration was observed at low frequency, but when sulfasalazine and dyclonine were administered in combination, high levels of intracellular HNE accumulation were observed at high frequency.

[0076] Thus, the combined use of an xCT inhibitor and an ALDH inhibitor frequently resulted in the accumulation of high levels of intracellular HNE. As mentioned above, multiple pathways exist for the degradation of HNE within cells (see Figure 12). The simultaneous inhibition of both the GST-mediated and ALDH-mediated pathways is thought to result in intracellular HNE accumulation. Furthermore, because HNE is cytotoxic, it is thought to prevent tumor cells from proliferating.

[0077] (Experimental Example 7) Effect of combined use of sulfasalazine or BSO with dyclonine analogues (those having a dyclonine skeleton) (Purpose) The following dyclonine analog (I) having a dyclonine skeleton is shown to have a combined effect with sulfasalazine or BSO.

[0078] JPEG2025114717000021.jpg43170 (in the formula, R 1 is a C1-6 linear or branched alkyl group, and R 2 and R 3are 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 ring with the N to which they are attached as a heteroatom, and R 4 is hydrogen or halogen. 1 is preferably a C4-5 linear or branched alkyl group, and R 2 and R 3 is a C2 alkyl group, or R 2 and R 3 are preferably taken together to form a 6-membered azacycloalkyl group with N as the hetero atom to which they are attached. 1 is a C4 linear alkyl group, and R 2 and R 3 The compound in which the groups taken together form a six-membered azacycloalkyl group with the N to which they are attached as a heteroatom is dyclonine. The halogen is preferably F, Cl, I, Br, or I. (Method) As in Experimental Example 1, HSC-4 cells were cultured in medium containing 0 μM (no additives), 25 μM, 50 μM, or 100 μM dyclonine, or 12.5 μM, 25 μM, 50 μM, or 100 μM of dyclonine analogs BAS00363846, STL327701, PHAR033081, PHAR298639, or Aldi-2 (see Figure 7B for structural formulas), and 0 μM (no additives), 100 μM BSO, or 300 μM sulfasalazine. Cell viability was measured and plotted in Figure 7A.

[0079] (Results) All of these compounds were effective when used in combination with BSO or sulfasalazine.

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

[0081] (Experimental Example 8) Effect of combined use of BSO and dyclonine analogs (not having a dyclonine skeleton) (Objective) To demonstrate that dyclonine analogs not having a dyclonine skeleton have no effect when used in combination with BSO.

[0082] (Method) As in Experimental Example 1, HSC-4 cells were cultured in medium containing 0 μM (no additive), 12.5 μM, 25 μM, or 50 μM dyclonine, or 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, or 100 μM dyclonine analog (4-hydroxyacetone: see Figure 8B for structural formula), and 0 μM (no additive) or 100 μM BSO, and cell viability was measured and graphed in Figure 8A.

[0083] (Results) Dyclonine analogs that do not have the dyclonine skeleton did not show any combined effects with BSO.

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

[0085] (Experimental Example 9) Effect of combined use of sulfasalazine, erastin, or BSO with dyclonine in sulfasalazine-resistant OSC19 cells (Objective) We demonstrate that dyclonine has a combined effect with glutathione synthesis inhibitors in cancer cell lines that have acquired resistance to xCT inhibitors.

[0086] (Methods) 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 OSC19 cell line 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 sulfasalazine, elastin, or BSO at the concentrations shown in Figure 9, plus 50 μM dyclonine (solvent: DMSO) or an equivalent amount of DMSO, and cultured for 48 hours. Cell viability was then measured using Celltiter-Glo (Promega). Cell viability was calculated relative to the control (without sulfasalazine, elastin, or BSO, but with DMSO) as 100%.

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

[0088] Thus, dyclonine exhibits a combined effect with glutathione synthesis inhibitors 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 cells (Objective) We demonstrate that the ALDH gene family is highly expressed in cancer cells resistant to xCT inhibitors.

[0090] (Methods) Messenger cells were isolated from HSC-4 cells, OSC19 cells, and OSC19-SSZR cells. The resulting complementary DNA was used as a template for quantitative RT-PCR to identify ALDHIAl and ALDHAI. The expression levels of ALDHIBl, ALDH2, ALDH3Al, and RPS17 were measured. Using the expression level of RPS17 as a reference, the expression level of each ALDH family gene was quantified by the ΔΔCt method, and the results are shown in FIG.

[0091] (Results) ALDHIAl expression was elevated in OSC19-SSZR compared to OSC19. High expression of ALDHIBl and ALDH2 was observed in HSC-4. High expression of ALDH3Al was observed in HSC4 and OSC19-SSZR. Thus, the expression of ALDH family genes tended to be high in cancer cell lines with low sensitivity to xCT.

[0092] Thus, in cancer cells with high expression of ALDH family genes, HNE is degraded by the ALDH family genes, and even if degradation to GST is suppressed by an xCT inhibitor, the toxicity of HNE does not occur, and the cells acquire resistance to the xCT inhibitor (see Figure 12). When such cells are administered an ALDH inhibitor, their sensitivity to the xCT inhibitor increases, so antitumor agents containing an ALDH inhibitor and a glutathione concentration-lowering agent or glutathione S-transferase inhibitor are effective even in cancer cells with high expression of ALDH family genes.

[0093] (Experimental Example 11) Effect of combined use of oxyfedrine (OXY) with sulfasalazine (SSZ) or L-buthionine sulfoximine (BSO) (Purpose) This experiment demonstrates that the combined use of sulfasalazine or L-buthionine-sulfoximine with oxyfedrine is effective in reducing the viability of sulfasalazine- or L-buthionine-sulfoximine-resistant tumor cells (A549, HCT116, HSC-4).

[0094] (Method) Sulfasalazine- or L-buthionine sulfoximine-resistant cell lines, including the lung basal adenocarcinoma cell line A549, colon adenocarcinoma cell line HCT116, and oral squamous cell carcinoma cell line HSC-4, were seeded at 4000 cells / well in 96-well plates. Culture medium was RPMI for A549, and DMEM for HCT116 and HSC-4. After 24 hours, the medium was replaced with medium containing 50 μM or 100 μM oxyfedrine or an equal volume of DMSO, plus 0 μM (no sulfasalazine or L-buthionine sulfoximine), 400 μM sulfasalazine, or 100 μM L-buthionine sulfoximine, and the culture was continued for 48 hours. The cell viability was then measured using Celltiter-Glo (Promega), and the cell viability for each case was calculated, with the number of viable cells in the control (DMSO added, no sulfasalazine or L-buthionine sulfoximine added) set at 100%. Figure 11 shows a graph showing the viability for each concentration of oxyfedrine.

[0095] (Results) 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. Furthermore, oxyfedrine alone (with oxyfedrine but without sulfasalazine) at 50 μM does not significantly kill the cells. However, when oxyfedrine and sulfasalazine or buthionine sulfoximine are both added, for example, at 100 μM oxyfedrine, viability drops to less than 25% with sulfasalazine and less than 5% with buthionine sulfoximine.

[0096] Thus, sulfasalazine or buthionine sulfoximine and oxyfedrine have a synergistic effect on the reduction of viability of sulfasalazine-resistant cells. However, the strength of this effect varies depending on the cell type. For example, considering administration to patients, a low concentration is preferable. For example, the combined effect of 50 μM oxyfedrine and sulfasalazine in A549 cells was almost the same as that in sulfasalazine alone, but in HCT116 cells, the cell viability was 75%, and in HSC-4 cells, it was also 75%. Thus, the combined effect of oxyfedrine and sulfasalazine in A549 cells was weaker than that in HCT116 cells and HSC-4 cells.

[0097] (Experimental Example 12) Reduction of GSH concentration in tumor cells by sulfasalazine (SSZ) or buthionine sulfoximine (BSO) (Objective) This experiment demonstrates that SSZ or BSO reduces GSH levels in tumor cells.

[0098] (Method) SSZ-resistant tumor cells (HCT116, HSC-4) 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 Figure 13.

[0099] (Results) When SSZ, BSO, or oxyfedrine (OXY) was administered alone, SSZ or BSO alone decreased intracellular GSH levels, but SSZ alone had little effect on decreasing intracellular GSH levels. OXY alone did not decrease intracellular GSH levels. On the other hand, when SSZ and OXY were administered together, a greater decrease in intracellular GSH levels was observed than when SSZ was administered alone.

[0100] Thus, SSZ or BSO function as xCT inhibitors and reduce GSH levels in tumor cells, but this effect is not sufficient when administered alone.

[0101] (Experimental Example 13) Accumulation of HNE by combined use of sulfasalazine (SSZ) or buthionine sulfoximine (BSO) with oxyfedrine (OXY) (Objective) This experiment demonstrates that the combined use of SSZ or BSO with OXY significantly increases the level of HNE in tumor cells and the frequency of cells that accumulate HNE.

[0102] (Method) In this experiment, 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 image observed under the fluorescence microscope is shown in Figure 14.

[0103] (Results) When SSZ, BSO, or OXY was treated alone, cells with increased intracellular HNE concentrations were observed at low frequency, but when SSZ or BSO was administered in combination with OXY, cells with high levels of intracellular HNE accumulation were observed at high frequency.

[0104] Thus, by using an xCT inhibitor and an ALDH inhibitor in combination, cells with high levels of intracellular HNE accumulation were frequently observed, suggesting that the combined use of an xCT inhibitor and an ALDH inhibitor has a synergistic effect on the reduction in viability of sulfasalazine-resistant cells, as in Example 11.

[0105] (Experimental Example 14) In vivo effects of combined use of sulfasalazine (SSZ) and oxyfedrine (OXY) (Purpose) This experiment demonstrates that the combined effects of SSZ and OXY can be observed in vivo.

[0106] (Method) Using HCT-116 cells, an oral squamous cell carcinoma cell line that is SSZ-resistant, tumors were formed in mice in the same manner as in Example 4, and the tumor volume (7 and 14 days after transplantation) and tumor weight (16 days after transplantation) were calculated, and the results were graphed in Figure 15. Photographs of the tumors were taken at the time of tumor weight measurement.

[0107] (Results) As shown in FIG. 15, each drug alone had almost no inhibitory effect on tumor growth, but the combined use of SSZ and OXY significantly inhibited tumor growth.

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

[0109] (Experimental Example 15) HNE accumulation in tumors in vivo by combined use of sulfasalazine (SSZ) and oxyfedrine (OXY) (Objective) In this experiment, we demonstrate 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) Tumors were formed in mice using the oral squamous cell carcinoma cell line HCT-116, an SSZ-resistant cell line, as in Example 4. 16 days after implantation, the tumors were subjected to immunohistochemical analysis as follows. First, tumor tissues were fixed in a 4% formaldehyde solution, and paraffin sections were prepared. Cell membrane permeabilization was performed with 0.2% TritonX100-PBS, followed by washing with PBS and blocking with 3% BSA-PBS. HNE was then stained brown using a Vectastain Elite kit (Vector Laboratories) with an anti-HNE antibody as the primary antibody and ImmPACT DAB Peroxidase Substrate (Vector Laboratories) as the enzyme substrate. Microscopic images are shown in Figure 16.

[0111] (Results) As shown in Figure 16, when SSZ and OXY were used in combination, cells with high levels of intracellular HNE were frequently observed in in vivo tumors. This suggests that, as in Example 14, the combined use of SSZ and OXY exerts a synergistic effect in suppressing the growth of tumors derived from SSZ-resistant cells.

[0112] Thus, the combined use of an xCT inhibitor and an ALDH inhibitor exerts a synergistic effect in suppressing the growth of tumors derived from sulfasalazine-resistant cells.

[0113] (Experimental Example 16) Effect of using ALDH inhibitors in tumor treatment with radiation (Purpose) In this experimental example, we demonstrate that by irradiating sulfasalazine (SSZ)-resistant cells with radiation and using an ALDH inhibitor at the same time, radiation exposure and the ALDH inhibitor have a synergistic effect on the reduction of viability and accumulation of HNE in SSZ-resistant cells.

[0114] (Method) In this experimental example, SSZ-resistant tumor cells (HCT116, HSC-4) were irradiated with 4, 6, or 10 Gy of ionizing radiation in the presence of 50 μM oxyfedrine (OXY) using an X-ray irradiator (Hitachi MBR-1520R-4, set at 150 kV, 20 mA). Control samples without OXY and samples not exposed to ionizing radiation (0 Gy) were also treated simultaneously. After 24 hours, cell viability was calculated as in Experimental Example 1. The results are shown in Figure 17. Intracellular HNE was visualized as in Example 6. The results are shown in Figure 18.

[0115] (Results) As shown in FIG. 17, the cell survival rate was significantly and remarkably reduced when radiation was irradiated in the presence of OXY, compared with when radiation was irradiated or OXY was treated alone. Furthermore, as shown in FIG. 18, cells with high intracellular HNE accumulation were observed at a high frequency and at a high concentration when irradiation treatment and OXY treatment were combined.

[0116] Thus, it is thought that the combined use of radiation and OXY treatment results in the accumulation of high levels of intracellular HNE, which significantly reduces cell viability. (Experimental Example 17) Correlation between Nrf2 expression level and xCT and ALDH expression levels (Purpose) This experimental example demonstrates that the expression level of Nrf2 is positively correlated with the expression levels of xCT and ALDH.

[0117] (Method) Expression of Nrf2, xCT, and β-actin was detected by Western blotting using extracts from SSZ-resistant tumor cells (HCT116, HSC-4, A549) using primary antibodies against Nrf2, xCT, and β-actin and HRP-conjugated secondary antibodies. Chemiluminescence reagent Plus (Perkin-Elmer Japan) was used for detection. The results are shown in Figure 19(A). Next, Nrf2 gene expression was suppressed by lipofecting A549 cells with siRNA against the Nrf2 gene. After 48 hours, extracts from the lipofected A549 cells were obtained and similarly subjected to Western blotting to examine the expression of Nrf2, xCT, ALDH3A1, and β-actin. The following sequence (bases in lowercase letters are DNA overhangs) was used as the siRNA. 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 Figure 19(B).

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

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

[0120] (Experimental Example 18) Effect of combined use of siRNA, sulfasalazine (SSZ) or buthionine sulfoximine (BSO), and oxyfedrine (OXY) on the Nrf2 gene (Objective) Figure 11 shows that the combined effect of oxyfedrine and sulfasalazine in A549 is weaker than that in HCT116 or HSC4. This experimental example demonstrates that suppressing the Nrf2 gene enhances the combined effect in A549.

[0121] (Method) A549 cells were transfected with siRNA against the Nrf2 gene as in Example 17, or the Nrf2 inhibitor ML385 was added to the medium. After 48 hours of culture in medium containing 50 μM OXY and either 400 μM SSZ or 100 μM BSO, cell viability was measured as in Example 11. The results are shown in Figure 20. As a control, experiments were performed using medium containing neither of these compounds, but DMSO was added appropriately so that the amount of DMSO added was constant in all cases.

[0122] (Results) As shown in Figure 20, in A549 cells, in addition to SSZ+OXY, siRNA against the Nrf2 gene or ML385, an Nrf2 inhibitor, can effectively reduce cell viability.

[0123] Thus, Nrf2 gene expression can be used as an indicator of whether co-administration of SSZ+OXY is effective in suppressing tumor growth. Furthermore, the effect of SSZ+OXY can be enhanced by administering siRNA against Nrf2 or ML385 in addition to SSZ+OXY. This strategy is particularly effective against tumors in which the Nrf2 gene is overexpressed. Example 19: Effect of using ALDH inhibitors in tumor treatment with radiation (Purpose) In this experimental example, by irradiating tumor cells transplanted into nude mice with radiation and using an ALDH inhibitor, we demonstrate that radiation and an ALDH inhibitor have a synergistic effect on the reduction of survival rate of SSZ-resistant cells and the accumulation of HNE. (Methods) 1.5x10 oral squamous cell carcinoma cell line HSC-2, a sulfasalazine-resistant cell line, was cultured in a 1000-well plate. 6The tumors were subcutaneously implanted into nude mice (n = 5). Oxyfedrine (OXY) (20 mg / kg) was administered intraperitoneally for three days (Days 1-3) starting the day after implantation. Oxyfedrine (30 mg / kg) was administered intraperitoneally on Day 4, and the nude mice were irradiated with X-rays (4, 6, or 10 Gy) two hours later. Oxyfedrine (20 mg / kg) was then administered intraperitoneally for three more days (Days 5-7). On Day 7, tumors were harvested and weighed. The results are shown in a bar graph in Figure 21. Nude mice were similarly implanted and administered oxyfedrine in the same manner but not irradiated with X-rays, and two groups were similarly irradiated with X-rays without oxyfedrine administration. Statistical analysis was performed using a Student's t-test between the oxyfedrine-treated and non-treated groups for each X-ray irradiation dose, with p<0.05 considered significant. (Results) As shown in Figure 21, tumor weight gain was suppressed as the X-ray intensity increased. In the non-X-ray irradiated group and the 4 Gy X-ray irradiated group, tumor weight gain tended to be suppressed in the oxyfedrine-administered group compared to the non-oxyfedrine-administered group, but in the 6 Gy X-ray irradiated group and the 10 Gy X-ray irradiated group, tumor weight gain was significantly suppressed in the oxyfedrine-administered group compared to the non-oxyfedrine-administered group. Thus, radiation exposure and oxyfedrine administration have a synergistic effect on the inhibition of tumor weight gain in vivo. [Industrial Applicability]

[0124] The present invention makes it possible to provide novel antitumor agents and combination agents.

Claims

1. An antitumor agent comprising, as an active ingredient, a glutathione concentration-lowering 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 pharmacologically acceptable salt thereof: (In the formula, R ５ is a C1-6 linear or branched alkyl group, and R ６ is hydrogen or halogen, and R ７ is a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl; R ８ is hydrogen or halogen.)

2. 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 administered simultaneously with an effective amount of a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor: (In the formula, R ５ is a C1-6 linear or branched alkyl group, and R ２ and R ３ are independently selected C1-6 straight or branched alkyl groups, or R ２ and R ３ together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group having N as a heteroatom to which they are attached, and R ４ is hydrogen or halogen.)

3. The antitumor agent according to claim 1 or 2, wherein the glutathione concentration-lowering agent is a drug that inhibits the activity of any one of xCT, thioredoxin-1 (TRX-1), glutamate-cysteine ligase (GCL) (EC 6.3.2.2) (also known as γ-glutamylcysteine synthetase), and glutathione synthetase (EC 6.3.2.3).

4. The antitumor agent according to claim 3, wherein the agent is an inhibitor of xCT or GCL.

5. The antitumor agent of claim 4, wherein the agent is sulfasalazine or L-buthionine sulfoximine.

6. The antitumor agent according to any one of claims 1 to 5, wherein the compound represented by formula (II) is oxyfedrine.

7. A combination drug containing, as active ingredients, an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof, and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor. (In the formula, R ５ is a C1-6 linear or branched alkyl group, and R ６ is hydrogen or halogen, and R ７ is a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl; R ８ is hydrogen or halogen.)

8. 8. The combination preparation according to claim 7, wherein the glutathione concentration-lowering agent is a drug that inhibits the activity of any one of xCT, thioredoxin-1 (TRX-1), glutamate-cysteine ligase (GCL) (EC 6.3.2.2) (also known as γ-glutamylcysteine synthetase), and glutathione synthetase (EC 6.3.2.3).

9. 9. The combination of claim 8, wherein the drug is an inhibitor of xCT or GCL.

10. 10. The combination of claim 9, wherein the drug is sulfasalazine or L-buthionine sulfoximine.

11. 11. The combination formulation of claim 10, wherein the compound represented by formula (II) is oxyfedrine.

12. An antitumor agent comprising the combination agent according to any one of claims 7 to 11.

13. The antitumor agent according to any one of claims 1 to 6 and 12, which is an antitumor agent for a tumor containing tumor cells resistant to a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor.

14. The antitumor agent according to claim 13, wherein aldehyde dehydrogenase is highly expressed in the tumor cells.

15. The antitumor agent according to claim 13 or 14, wherein the tumor further comprises tumor cells expressing CD44v.

16. Simultaneously administering an aldehyde dehydrogenase inhibitor or compound (III) or a pharmacologically acceptable salt thereof and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor to tumor cells in vitro; and measuring the proliferation rate or cell survival rate of the tumor cells. (wherein X is hydrogen, halogen, —NH ２ , or —CN, Y is a C1-6 linear or branched alkyl group, Z １ and Z ２ are each hydrogen or halogen, and an optionally substituted C1-6 linear or branched alkyl group, the substituent being hydroxy or phenyl, or Z １ and Z ２ are taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom.

17. A method for identifying a drug having a combined effect with a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, comprising: Simultaneously administering a specific glutathione concentration-lowering agent or glutathione S-transferase inhibitor and a plurality of aldehyde dehydrogenase inhibitors or compound (III) or a pharmacologically acceptable salt thereof to tumor cells in vitro; and measuring the proliferation rate or cell viability of the tumor cells. (wherein X is hydrogen, halogen, —NH ２ , or —CN, Y is a C1-6 linear or branched alkyl group, Z １ and Z ２ are each hydrogen or halogen, and an optionally substituted C1-6 linear or branched alkyl group, the substituent being hydroxy or phenyl, or Z １ and Z ２ are taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom.

18. A method for identifying a glutathione concentration-lowering agent or a glutathione-S-transferase inhibitor having a combined effect with an antitumor agent, compound (III) or a pharmacologically acceptable salt thereof, comprising: administering compound (III) and a plurality of glutathione concentration-lowering agents or glutathione S-transferase inhibitors simultaneously to tumor cells in vitro; and measuring the proliferation rate or cell viability of the tumor cells. (wherein X is hydrogen, halogen, —NH ２ , or —CN, Y is a C1-6 linear or branched alkyl group, Z １ and Z ２ are each hydrogen or halogen, and an optionally substituted C1-6 linear or branched alkyl group, the substituent being hydroxy or phenyl, or Z １ and Z ２ are taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom.

19. The method for identifying compound (III) according to claim 17 or 18, wherein compound (III) is compound (II). (In the formula, R ５ is a C1-6 linear or branched alkyl group, and R ２ and R ３ are independently selected C1-6 straight or branched alkyl groups, or R ２ and R ３ together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group having N as a heteroatom to which they are attached, and R ４ is hydrogen or halogen.)

20. A method for identifying tumor cells that exhibits the combined effect of an antitumor agent, compound (III) or a pharmacologically acceptable salt thereof, and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, comprising: Simultaneously administering a specific combination of compound (III) or a pharmacologically acceptable salt thereof and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor to a plurality of tumor cells in vitro; and measuring the proliferation rate or cell viability of said plurality of tumor cells. (wherein X is hydrogen, halogen, —NH ２ , or —CN, Y is a C1-6 linear or branched alkyl group, Z １ and Z ２ are each hydrogen or halogen, and an optionally substituted C1-6 linear or branched alkyl group, the substituent being hydroxy or phenyl, or Z １ and Z ２ are taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom.

21. The method of claim 20, wherein the compound (III) is compound (II). (In the formula, R ５ is a C1-6 linear or branched alkyl group, and R ２ and R ３ are independently selected C1-6 straight or branched alkyl groups, or R ２ and R ３ together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group having N as a heteroatom to which they are attached, and R ４ is hydrogen or halogen.)

22. The method of claim 16, wherein the tumor cells are resistant to glutathione concentration-lowering agents or glutathione S-transferase inhibitors.

23. The method for identifying tumor cells according to any one of claims 17 to 21, wherein the tumor cells are resistant to a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor.

24. 1. An antitumor agent for use in radiation therapy, which contains, as an active ingredient, an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof: (In the formula, R ５ is a C1-6 linear or branched alkyl group, and R ２ and R ３ are independently selected C1-6 straight or branched alkyl groups, or R ２ and R ３ together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group having N as a heteroatom to which they are attached, and R ４ is hydrogen or halogen.)

25. The radiation-combined antitumor agent according to claim 24, wherein the compound represented by formula (II) is oxyfedrine.

26. The antitumor agent combined with radiation according to claim 24 or 25, which is an antitumor agent for a tumor containing tumor cells resistant to a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor.

27. The antitumor agent combined with radiation according to claim 26, wherein aldehyde dehydrogenase is highly expressed in the tumor cells.

28. The antitumor agent combined with radiation according to claim 26 or 27, wherein the tumor further comprises tumor cells expressing CD44v.

29. irradiating tumor cells in vitro in the presence of an aldehyde dehydrogenase inhibitor or compound (III) or a pharmaceutically acceptable salt thereof; and measuring the proliferation rate or cell survival rate of the tumor cells. (wherein X is hydrogen, halogen, —NH ２ , or —CN, Y is a C1-6 linear or branched alkyl group, Z １ and Z ２ are each hydrogen or halogen, and an optionally substituted C1-6 linear or branched alkyl group, the substituent being hydroxy or phenyl, or Z １ and Z ２ are taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom.

30. The method for measuring an antitumor effect according to claim 29, wherein the tumor cells are resistant to a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor.

31. 1. A method for identifying an agent that has a synergistic effect with radiation in tumor cells in vitro, comprising: Irradiating tumor cells in vitro in the presence of each of multiple aldehyde dehydrogenase inhibitors or compound (III) or a pharmacologically acceptable salt thereof; and measuring the proliferation rate or cell viability of the tumor cells. (wherein X is hydrogen, halogen, —NH ２ , or —CN, Y is a C1-6 linear or branched alkyl group, Z １ and Z ２ are each hydrogen or halogen, and an optionally substituted C1-6 linear or branched alkyl group, the substituent being hydroxy or phenyl, or Z １ and Z ２ are taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group with the N to which they are attached as a heteroatom.

32. The method of claim 31, wherein compound (III) is compound (II). (In the formula, R ５ is a C1-6 linear or branched alkyl group, and R ２ and R ３ are independently selected C1-6 straight or branched alkyl groups, or R ２ and R ３ together form a 4-, 5-, 6-, or 7-membered azacycloalkyl group having N as a heteroatom to which they are attached, and R ４ is hydrogen or halogen.)

33. The method of claim 31 or 32, wherein the tumor cells are resistant to glutathione concentration-lowering agents or glutathione S-transferase inhibitors.

34. An antitumor effect enhancer obtained by co-administration of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof with a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, the antitumor effect enhancer containing an inhibitor of the xCT expression enhancing effect of Nrf2. (In the formula, R ５ is a C1-6 linear or branched alkyl group, and R ６ is hydrogen or halogen, and R ７ is a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl; R ８ is hydrogen or halogen.)

35. The antitumor effect enhancer according to claim 34, wherein the inhibitor is an expression inhibitor of the Nrf2 gene or an Nrf2 inhibitor.

36. The antitumor effect enhancer described in claim 35, wherein the expression inhibitor against the Nrf2 gene is an antisense NA, miNA, or siNA against the Nrf2 gene.

37. The anti-tumor effect enhancer according to claim 35, wherein the Nrf2 inhibitor is ML385 or an anti-Nrf2 antibody.

38. The antitumor effect enhancer according to any one of claims 34 to 37, wherein the antitumor effect is an effect on tumors that overexpress the Nrf2 gene.

39. The antitumor effect enhancer according to any one of claims 34 to 38, wherein the glutathione concentration lowering agent is sulfasalazine.

40. The antitumor agent according to any one of claims 34 to 39, wherein the compound represented by formula (II) is oxyfedrine.

41. A companion diagnostic agent for predicting an antitumor effect of coadministration of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof with a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, comprising: A companion diagnostic agent comprising a reagent for detecting Nrf2 gene expression. (In the formula, R ５ is a C1-6 linear or branched alkyl group, and R ６ is hydrogen or halogen, and R ７ is a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl; R ８ is hydrogen or halogen.)

42. The companion diagnostic agent according to claim 41 , wherein the detection reagent is an antibody, a probe for detecting gene expression, or a primer for amplifying a gene.

43. The companion diagnostic agent for an antitumor effect according to claim 41 or 42, wherein the glutathione concentration lowering agent is sulfasalazine.

44. The companion diagnostic agent according to any one of claims 41 to 43, wherein the compound represented by formula (II) is oxyfedrine.

45. A companion diagnostic agent for predicting an antitumor effect of coadministration of an aldehyde dehydrogenase inhibitor or the following compound (II) or a pharmacologically acceptable salt thereof with a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, comprising: A companion diagnostic agent comprising a reagent for detecting mutations in the Keap1 gene or the Nrf2 gene. (In the formula, R ５ is a C1-6 linear or branched alkyl group, and R ６ is hydrogen or halogen, and R ７ is a C1-6 linear or branched alkyl group which may have a substituent, and the substituent is hydroxy or phenyl; R ８ is hydrogen or halogen.)

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