Antitumor agent and combination drug

Abstract

To provide a new antitumor agent.SOLUTION: The present invention provides an antitumor agent containing a glutathione concentration lowering agent or a glutathione S-transferase inhibitor as an active ingredient to be administered simultaneously with an aldehyde dehydrogenase inhibitor, or an antitumor agent containing an aldehyde dehydrogenase inhibitor as an active ingredient to be administered simultaneously with an effective amount of a glutathione concentration lowering agent or a glutathione S-transferase inhibitor, or a combination drug containing an aldehyde dehydrogenase inhibitor and a glutathione concentration lowering agent or a glutathione S-transferase inhibitor as active ingredients. The aldehyde dehydrogenase inhibitor is a compound represented by the following formula (I) or a pharmacologically acceptable salt thereof.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 traits, 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 sulfasalazine with an aldehyde dehydrogenase inhibitor has a significant antitumor effect against tumor cells to which sulfasalazine 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 containing, 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 an antitumor agent containing, as an active ingredient, an aldehyde dehydrogenase inhibitor, which is administered simultaneously with an effective amount of a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor.

[0010] Another embodiment of the present invention is a combination drug containing an aldehyde dehydrogenase inhibitor and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor as active ingredients.

[0011] A further embodiment of the present invention is an antitumor agent containing the above combination agent.

[0012] The glutathione concentration-lowering agent may be a drug that inhibits the activity of any 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). The drug may be an xCT transporter inhibitor. The xCT transporter inhibitor may be sulfasalazine, erastin, or sorafenib. The aldehyde dehydrogenase inhibitor may be a compound represented by the following formula (I) or a pharmacologically acceptable salt thereof.

[0013] JPEG2026001113000001.jpg30121 (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 4 is hydrogen or halogen.) A further embodiment of the present invention 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-, or 6-membered azacycloalkyl group with the N to which they are attached as a heteroatom. The compound represented by formula (I) may be Dyclonine, BAS00363846, STL327701, PHAR033081, PHAR298639, or Aldi-2.

[0014] A further embodiment of the present invention is a measurement method comprising the steps of simultaneously administering an aldehyde dehydrogenase inhibitor 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.

[0015] A further embodiment of the present invention is a method for identifying an aldehyde dehydrogenase inhibitor that has 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 multiple aldehyde dehydrogenase inhibitors to tumor cells in vitro, and measuring the proliferation rate or cell viability of the tumor cells.

[0016] A further embodiment of the present invention is a method for identifying a glutathione concentration-lowering agent or glutathione S-transferase inhibitor that has a combined effect with an aldehyde dehydrogenase inhibitor, the method comprising the steps of simultaneously administering a specific aldehyde dehydrogenase inhibitor that is an antitumor agent and multiple 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.

[0017] A further embodiment of the present invention is a method for identifying tumor cells that benefit from the combined effect of an aldehyde dehydrogenase inhibitor and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, the method comprising the steps of simultaneously administering a specific combination of an aldehyde dehydrogenase inhibitor and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor to multiple tumor cells in vitro, and measuring the proliferation rate or cell viability of the multiple tumor cells.

[0018] In any of the above-mentioned methods for identifying or measuring, the tumor cells may be resistant to a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor.

[0019] ==Cross-reference to related literature== This application claims priority based on Japanese Patent Application No. 2017-220231, filed on November 15, 2017, and the basic application is incorporated herein by reference. [Brief explanation of the drawings]

[0020] [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 effect of combined use of sulfasalazine, erastin, or BSO with dyclonine in 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] Figure 1 shows the metabolic pathway of HNE. Abbreviations: HNA, 4-hydroxy-2-nonenoic acid; GSH, glutathione; ALDH, aldehyde dehydrogenase; GST, glutathione S-transferase. DETAILED DESCRIPTION OF THE INVENTION

[0021] 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.

[0022] 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.

[0023] ==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, wherein the effective amount of the aldehyde dehydrogenase inhibitor is an amount that has a combined antitumor effect with the glutathione concentration-lowering agent.

[0024] Another embodiment of the present invention is an antitumor agent containing an aldehyde dehydrogenase inhibitor as an active ingredient, which is administered simultaneously with an effective amount of a glutathione concentration-lowering agent, wherein 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.

[0025] 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.

[0026] JPEG2026001113000002.jpg30121 (in the formula, R 1is 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. 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. Specific examples of pharmacologically acceptable salts include addition salts with inorganic acids such as hydrochloride, sulfate, nitrate, hydrobromide, hydroiodide, perchlorate, and phosphate; addition salts with 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.

[0027] Glutathione concentration-lowering agents are drugs that reduce intracellular glutathione concentrations. While the glutathione concentration-lowering agents used in these antitumor agents are not limited, drugs that inhibit the pathway by which glutathione is produced from cystine taken up into cells by xCT are preferred. Drugs 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 being more preferred. While the xCT inhibitor is not particularly limited, sulfasalazine, erastin, sorafenib, or an anti-xCT antibody is preferred.

[0028] 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.

[0029] Here, "simultaneous administration" of two drugs means not only administration at the same time, but also administration of each drug separately at different times, as long as the effect of one drug remains while the other drug is still being administered. When administering two drugs simultaneously, two drugs containing only one drug may be administered at the same time, or the two drugs may be administered in a single dosage form as a combination drug.

[0030] 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 0-24 Sulfasalazine-resistant tumor cells are tumor cells that survive administration of 50 to 300 μg h / mL for approximately two weeks, and have a survival rate of 90% or more at 200 μM in vitro. Sulfasalazine-resistant tumor cells preferably also have low levels of CD44v expression or are negative. Tumor cells overexpressing aldehyde dehydrogenase are cells expressing any of the genes ALDH1A1, ALDH2, ALDH1B1, and ALDH3A1 at levels three times or more, preferably ten times or more, higher than those of OSC19 cells. The tumor to be treated may contain tumor cells expressing CD44v. This is because sulfasalazine has 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 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.

[0031] 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.

[0032] The antitumor agent may be formulated into tablets, powders, granules, powders, 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.

[0033] 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.

[0034] ==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 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 to which the agents have been administered. The aldehyde dehydrogenase inhibitor, glutathione concentration-lowering agent, and glutathione S-transferase inhibitor in this section are the same as those described in detail in the "Antineoplastic Agent" section.

[0035] Aldehyde dehydrogenase inhibitors and glutathione concentration-lowering agents or glutathione S-transferase inhibitors have a combined effect on antitumor activity, so this measurement method can be used to identify drug combinations that have a high combined effect or to identify tumor cells that are particularly effective against a certain drug combination.

[0036] Specifically, by simultaneously administering a specific glutathione concentration-lowering agent or glutathione S-transferase inhibitor and multiple aldehyde dehydrogenase inhibitors to tumor cells in vitro and measuring the proliferation rate or cell viability of the drug-administered tumor cells, it is possible to identify aldehyde dehydrogenase inhibitors that have a combined effect with a specific glutathione concentration-lowering agent or glutathione S-transferase inhibitor.Furthermore, by simultaneously administering a specific aldehyde dehydrogenase inhibitor and multiple glutathione concentration-lowering agents or glutathione S-transferase inhibitors to tumor cells in vitro and measuring the proliferation rate or cell viability of the drug-administered tumor cells, it is possible to identify glutathione concentration-lowering agents or glutathione S-transferase inhibitors that have a combined effect with a specific aldehyde dehydrogenase inhibitor. Alternatively, by simultaneously administering a specific combination of an aldehyde dehydrogenase inhibitor and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor to multiple tumor cells in vitro and measuring the proliferation rate or cell viability of multiple tumor cells to which the drugs have been administered, it is possible to identify tumor cells that benefit from the combined effects of an aldehyde dehydrogenase inhibitor and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor. [Example]

[0037] (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.

[0038] (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.

[0039] (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.

[0040] Thus, the combined use of sulfasalazine and dyclonine is effective in reducing the viability of sulfasalazine-resistant cells.

[0041] (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.

[0042] (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.

[0043] (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.

[0044] Thus, the combined effect of sulfasalazine and dyclonine is mediated by the xCT inhibitory effect of sulfasalazine.

[0045] (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.

[0046] (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.

[0047] (Results) Although the effect varied depending on the cell type, similar combined effects were observed with sulfasalazine, erastin, or BSO and dyclonine.

[0048] 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.

[0049] (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.

[0050] (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.

[0051] Tumor volume = (longer diameter x (short diameter) 2 ) / 2 Statistical analysis of tumor volume was performed on day 22 using t-test.

[0052] (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%.

[0053] Thus, combined administration of sulfasalazine and dyclonine can reduce the growth of sulfasalazine-resistant tumors.

[0054] (Experimental Example 5) Inhibition of ALDH by Dyclonine (Objective) This experimental example demonstrates that dyclonine has ALDH inhibitory activity.

[0055] (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 positive cells were 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.

[0056] (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%.

[0057] Thus, dyclonine has inhibitory activity against ALDH.

[0058] (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.

[0059] (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.

[0060] (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.

[0061] Thus, the combined use of an xCT inhibitor and an ALDH inhibitor frequently resulted in the accumulation of high levels of intracellular HNE. While not being bound by the following theory, the reason for this is thought to be that, as shown in Figure 11, there are multiple pathways for HNE degradation within cells, and by simultaneously inhibiting both the GST-mediated and ALDH-mediated degradation pathways, HNE accumulates within the cells. Furthermore, because HNE is cytotoxic, it is thought that tumor cells are unable to proliferate.

[0062] (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.

[0063] JPEG2026001113000003.jpg30121 (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.

[0064] (Results) All of these compounds were effective when used in combination with BSO or sulfasalazine.

[0065] Thus, the dyclonine analog (I) having a dyclonine skeleton has a combined effect as an xCT inhibitor and an antitumor agent.

[0066] (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.

[0067] (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.

[0068] (Results) Dyclonine analogs that do not have the dyclonine skeleton did not show any combined effects with BSO.

[0069] Thus, the dyclonine skeleton is important for interaction with xCT inhibitors.

[0070] (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.

[0071] (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%.

[0072] (Results) Dyclonine also showed a combined effect with sulfasalazine, erastin, or BSO in OSC19-SSZR cells.

[0073] Thus, dyclonine exhibits a combined effect with glutathione synthesis inhibitors even in cancer cells that have acquired resistance to xCT inhibitors.

[0074] (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.

[0075] (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.

[0076] (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.

[0077] 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 11). 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-reducing agent or glutathione S-transferase inhibitor are effective even in cancer cells with high expression of ALDH family genes. [Industrial Applicability]

[0078] The present invention makes it possible to provide novel antitumor agents and combination agents.

Claims

1. An antitumor agent containing 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.

2. An antitumor agent containing an aldehyde dehydrogenase inhibitor as an active ingredient, which is administered simultaneously with an effective amount of a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor.

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 the xCT transporter.

5. The antitumor agent according to claim 4, wherein the xCT transporter inhibitor is sulfasalazine, erastin, or sorafenib.

6. The antitumor agent according to any one of claims 1 to 5, wherein the aldehyde dehydrogenase inhibitor is a compound represented by the following formula (I) or a pharmacologically acceptable salt thereof: (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 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.)

7. The antitumor agent according to claim 6, wherein the compound represented by formula (I) is dyclonine, BAS00363846, STL327701, PHAR033081, PHAR298639, or Ald-2.

8. A combination drug containing an aldehyde dehydrogenase inhibitor and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor as active ingredients.

9. 9. The combination formulation of claim 8, wherein the glutathione concentration-lowering agent is an inhibitor of the xCT transporter.

10. 10. The combination of claim 9, wherein the inhibitor of the xCT transporter is sulfasalazine, erastin, or sorafenib.

11. The combination formulation according to any one of claims 8 to 10, wherein the aldehyde dehydrogenase inhibitor is a compound represented by formula (I) or a pharmacologically acceptable salt thereof. (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 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.)

12. 12. The combination formulation of claim 11, wherein the compound represented by formula (3) is dyclonine, BAS00363846, STL327701, PHAR033081, PHAR298639, or Ald-2.

13. An antitumor agent comprising the combination agent according to any one of claims 8 to 12.

14. The antitumor agent according to any one of claims 1 to 7 and 13, which is an antitumor agent for a tumor containing tumor cells resistant to a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor.

15. The antitumor agent according to claim 14, wherein the glutathione concentration-lowering agent is an xCT inhibitor.

16. The antitumor agent of claim 15, wherein the xCT inhibitor is sulfasalazine.

17. The antitumor agent according to any one of claims 14 to 16, wherein aldehyde dehydrogenase is highly expressed in the tumor cells.

18. The antitumor agent according to any one of claims 14 to 17, wherein the tumor further comprises tumor cells expressing CD44v.

19. co-administering an aldehyde dehydrogenase inhibitor 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.

20. A method for identifying an aldehyde dehydrogenase inhibitor having a combined effect with a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, comprising: co-administering a specific glutathione concentration-lowering agent or glutathione S-transferase inhibitor and multiple aldehyde dehydrogenase inhibitors to tumor cells in vitro; and measuring the proliferation rate or cell viability of the tumor cells.

21. A method for identifying a glutathione concentration-lowering agent or a glutathione-S-transferase inhibitor that has a combined effect with an aldehyde dehydrogenase inhibitor, comprising: A step of simultaneously administering a specific aldehyde dehydrogenase inhibitor, which is an antitumor agent, and multiple 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.

22. A method for identifying tumor cells that exhibit the combined effect of an aldehyde dehydrogenase inhibitor and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor, comprising: administering a specific combination of an aldehyde dehydrogenase inhibitor and a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor simultaneously to a plurality of tumor cells in vitro; and measuring the proliferation rate or cell viability of said plurality of tumor cells.

23. The method of claim 19, wherein the tumor cells are resistant to a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor.

24. The method for identifying tumor cells according to any one of claims 20 to 22, wherein the tumor cells are resistant to a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor.

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