WO2019098288A1

WO2019098288A1 - Antitumor agent and compounding agent

Info

Publication number: WO2019098288A1
Application number: PCT/JP2018/042327
Authority: WO
Inventors: 永野　修; 秀行佐谷; 章悟岡崎
Original assignee: 学校法人慶應義塾
Priority date: 2017-11-15
Filing date: 2018-11-15
Publication date: 2019-05-23
Also published as: JP2024023269A; JPWO2019098288A1; JP7388702B2

Abstract

Provided is: a novel antitumor agent which contains, as an active ingredient, a glutathione concentration-decreasing agent or a glutathione S-transferase inhibitor which is simultaneously administered with an effective amount of an aldehyde dehydrogenase inhibitor; or a novel antitumor agent which contains, as an active ingredient, an aldehyde dehydrogenase inhibitor which is simultaneously administered with an effective amount of a glutathione concentration-decreasing agent or a glutathione S-transferase inhibitor; or a novel compounding agent which contains, as active ingredients, an aldehyde dehydrogenase inhibitor and a glutathione concentration-decreasing agent or glutathione S-transferase inhibitor.

Description

Antineoplastic agent and combination agent

The present invention relates to antineoplastic agents and combinations.

In cancer treatment, the presence of cells resistant to treatments such as anti-cancer agents and radiation causes recurrence and metastasis, which hinders the treatment of cancer. Recently, attention has been focused on the presence of cancer stem cells as such therapeutic resistant cells. Cancer stem cells are highly resistant to various types of stress, and development of drugs targeting cancer stem cells is urgently needed to eradicate cancer. However, the analysis of the molecular mechanisms of cancer stem cell stress tolerance for the development of treatments targeting cancer stem cells has just begun.

CD44, which is one of the markers of epithelial cancer stem cells, is known as a molecule involved in stress resistance (Cancer Cell. 2011 Mar 8; 19 (3): 387-400). Splice variant forms (hereinafter, CD44v) exist in CD44, and CD44v stably expresses cystine transporter xCT on the cell membrane. Since xCT has a function of taking in cystine into cells, and the incorporated cystine is used for the production of glutathione (GSH), the amount of GSH is increased in cells highly expressing CD44v. Because GSH has a strong antioxidant action and plays a role in reducing cell-induced stress, cancer stem cells that highly express CD44v are considered to be resistant to treatment.

On the other hand, a drug used for treatment of ulcerative colitis and rheumatoid arthritis is sulfasalazine (alias: salazosulfapyridine, salazopyrine, salicylazosulfapyridine). Sulfasalazine is an acidic azo compound of sulfapyridine and 5-aminosalicylic acid (5-ASA), and when orally administered, it is decomposed into sulfapyridine and 5-aminosalicylic acid (5-ASA) by enterobacteria in the intestine Ru. In particular, 5-ASA is the main active ingredient for the above-mentioned diseases.

In recent years, the unaltered sulfasalazine before decomposition has been found to be effective as an antitumor agent, having xCT inhibitory activity (Leukemia vol. 15, pp. 1633-1640, 2001). That is, when sulfasalazine is added to cancer cells, uptake of cystine into cells by xCT is suppressed, glutathione production is reduced, and as a result, resistance to cancer cells is reduced by oxidative stress, and sensitivity to antitumor agents is increased. Do.

It is known that sulfasalazine, which has an xCT inhibitory action, effectively suppresses proliferation of cancer stem cells that highly express CD44v (Japanese Patent Laid-Open No. 2012-144498).

An object of the present invention is to provide novel antitumor agents and combination agents.

The present inventors have found that sulfasalazine alone has an antitumor effect on tumors in which undifferentiated tumor cells are mostly, but CD44v on differentiated tumors that include tumor cells exhibiting differentiation characteristics. It has been found that although it reduces cancer stem cells that overexpress, it has no effect on reducing the overall tumor volume. Therefore, for such differentiated tumors, we have made intensive efforts to obtain an antitumor agent for differentiated tumors by developing a drug having an antitumor effect for tumor cells in which sulfasalazine has no antitumor effect. When the aldehyde dehydrogenase inhibitor is used in combination with sulfasalazine, it was found that sulfasalazine alone has a remarkable antitumor effect on tumor cells having a weak effect, leading to the completion of the present invention.

One embodiment of the present invention is an antitumor agent containing a glutathione concentration reducing agent or a glutathione S-transferase inhibitor as an active ingredient, which is administered simultaneously with an effective amount of an aldehyde dehydrogenase inhibitor, or an effective amount And an antitumor agent containing an aldehyde dehydrogenase inhibitor as an active ingredient, which is administered simultaneously with the glutathione concentration-reducing agent or the glutathione S-transferase inhibitor.

Another embodiment of the present invention is a combination drug comprising an aldehyde dehydrogenase inhibitor and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor as active ingredients.

A further embodiment of the present invention is an antitumor agent containing the above-mentioned combination drug.

The above-mentioned glutathione concentration-lowering agent is xCT, Thioredoxin-1 (thioredoxin-1: TRX-1), glutamate-cysteine ligase (GCL) (EC 6.3.2.2) (also called γ-glutamylcysteine synthetase), glutathione synthesis It may be an agent that inhibits any activity of the enzyme (EC 6.3.2.3). The agent may be an inhibitor of the xCT transporter. The inhibitor of the xCT transporter may be sulfasalazine, elastin, or sorafenib. The aldehyde dehydrogenase inhibitor may be a compound represented by the following formula (I) or a pharmacologically acceptable salt thereof.

(Wherein, R ¹ is a C 1-6 linear or branched alkyl group, and R ² and R ³ are independently selected C 1-6 linear or branched alkyl groups, or R ² and R 3 ³ is taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group having the hetero atom N to which they are bonded, and R ⁴ is hydrogen or halogen. )
A further embodiment of the present invention is a straight-chain or branched alkyl group of C1 ~ 6, or R ² and R ³ is a linear or branched alkyl group of C1 ~ 6 are independently selected, and R ² R ^{3 taken} together form a 4-, 5-, or 6-membered azacycloalkyl group having the heteroatom N as the hetero atom to which they are attached. )
The compound represented by the formula (I) may be diclonine, BAS00363846, STL327701, PHAR033081, PHAR298639 or Aldi-2.

A further embodiment of the present invention comprises simultaneously administering an aldehyde dehydrogenase inhibitor and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor to tumor cells in vitro, and the growth rate or cell viability of the tumor cells. And a measuring step.

A further embodiment of the present invention is a method for identifying an aldehyde dehydrogenase inhibitor having a combined effect with a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, wherein the specific glutathione concentration reducing agent or a glutathione S-transferase inhibitor And simultaneously administering a plurality of aldehyde dehydrogenase inhibitors to tumor cells in vitro, and measuring the growth rate or cell viability of the tumor cells.

A further embodiment of the present invention is a method for identifying a glutathione concentration reducing agent or glutathione S-transferase inhibitor that has a combined effect with an aldehyde dehydrogenase inhibitor, wherein the specific aldehyde dehydrogenase inhibitor is an antitumor agent And simultaneously administering a plurality of glutathione concentration lowering agents or glutathione S-transferase inhibitors to tumor cells in vitro, and measuring the growth rate or cell viability of the tumor cells. is there.

A further embodiment of the present invention is a method of identifying a tumor cell which exerts a combined effect of an aldehyde dehydrogenase inhibitor and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, comprising: the aldehyde dehydrogenase inhibitor and glutathione concentration A specific method comprising simultaneously administering a specific combination of a hypotensive agent or a glutathione S-transferase inhibitor to a plurality of tumor cells in vitro, and measuring the growth rate or cell viability of the plurality of tumor cells It is.

In any of the above-mentioned specific methods or measurement methods, the tumor cell may be resistant to a glutathione concentration-reducing agent or a glutathione S-transferase inhibitor.

== Cross reference with related documents ==
This application claims priority based on Japanese Patent Application No. 2017-220231 filed on Nov. 15, 2017, which is incorporated herein by reference.

It is the graph which showed the combined effect of sulfasalazine and diclonine in one example of the present invention. In one Example of this invention, it is the graph which showed the change of the dicronine sensitivity by xCT knockdown. FIG. 7 is a view showing the combined effect of sulfasalazine, elastin, or BSO and diclonine in various cancer cell lines in one example of the present invention. In one Example of this invention, it is the graph which showed the in-vivo combined effect of sulfasalazine and diclonine. FIG. 6 is a diagram of experimental results showing the inhibitory effect of ALDH activity by dyclonine in one example of the present invention. FIG. 7 is a diagram showing the accumulation effect of HNE (4-HNE; 4-hydroxy-2-nonenal) by the combined use of sulfasalazine and diclonine in one example of the present invention. In one Example of this invention, it is the graph which showed the combined effect of sulfasalazine or BSO and a diclonine analog (those which have a diclonine frame | skeleton). In one Example of this invention, it is the graph which showed the combined effect of BSO and a diclonine analog (those which do not have a diclonine frame | skeleton). FIG. 6 is a graph showing the combined effect of sulfasalazine, elastin, or BSO and dyclonine in OSC19 cells or sulfasalazine-resistant OSC19 cells in one example of the present invention. In the Example of this invention, it is the graph which showed expression of the ALDH gene family in HSC4 cell, OSC19 cell, or sulfasalazine resistant OSC19 cell. It is a figure showing the metabolic pathway of HNE. Abbreviations: HNA, 4-hydroxy-2-nonenoic acid; GSH, glutathione; ALDH, aldehyde dehydrogenase; GST, glutathione S-transferase

Hereinafter, embodiments of the present invention will be described in detail by way of examples. The objects, features, advantages, and ideas of the present invention will be apparent to those skilled in the art from the description of the present specification, and those skilled in the art will readily reproduce the present invention from the descriptions of the present specification. it can. The embodiments and specific examples of the invention described below show preferred embodiments of the present invention and are shown for the purpose of illustration or explanation. It is not limited. It will be apparent to those skilled in the art that various changes and modifications can be made based on the description of the present specification within the intent and scope of the present invention disclosed herein.

In addition, when there is no particular description in the embodiment and the examples, the method described in the standard protocol collection, or a method obtained by modifying or modifying it is used. Moreover, when using a commercially available reagent kit or measuring apparatus, unless otherwise described, the protocol attached thereto is used.

== Antineoplastic agent = =
One embodiment of the present invention is an antitumor agent containing a glutathione concentration reducing agent as an active ingredient, which is administered simultaneously with an effective amount of an aldehyde dehydrogenase inhibitor. Here, the effective amount of the aldehyde dehydrogenase inhibitor is an aldehyde dehydrogenase inhibitor in an amount having a combined effect with a glutathione concentration reducing agent as an antitumor activity.

In addition, 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. Here, the effective amount of the glutathione concentration reducing agent is a glutathione concentration reducing agent having an effect in combination with an aldehyde dehydrogenase inhibitor as an antitumor activity.

Aldehyde dehydrogenase inhibitors are agents that inhibit the enzyme activity of aldehyde dehydrogenase 2 (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 ALDH 1-5 and their isotypes. Aldehyde dehydrogenase inhibitors used in antitumor agents are not particularly limited, but clopropamide (chlorpropamide), tolbutamide (tolbutamide), diethylaminobenzaldehyde, tetraethylthioperoxydicarbonic diamide, cyanamide (cyanamide), oxyphedrine, citral ( 3,7-dimethyl-2,6-octadienal), coprin (coprine), daidzin, DEAB (4- (Diethylamino) benzaldehyde), gossypol (gossypol), kynurenine metabolites (3-hydroxykynurenine, 3-hydroxycyanuranilic acid) , Kynurenic acid, and indol-3-ylpyruvic acid), molinate (Molinate), nitroglycerin, purgerin (N-benzyl-N-methylprop-2-yn-1-amine) and their analogs, or their pharmacology And salts acceptable for In particular, the following dicronine and diclonine analogues (I) are preferred, and the compounds shown in FIG. 7 (BAS00363846, STL327701, PHAR033081, PHAR298639, and Aldi-2) are more preferred.

(Wherein, R ¹ is a C 1-6 linear or branched alkyl group, and R ² and R ³ are independently selected C 1-6 linear or branched alkyl groups, or R ² and R 3 ³ is taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group having the hetero atom N to which they are bonded, and R ⁴ is hydrogen or halogen. R ¹ is preferably a C 4 to 5 linear or branched alkyl group, and R ² and R ³ are C 2 alkyl groups, or R ² and R ^{3 taken} together are the N to which they are bonded is preferably a 6-membered azacycloalkyl group ring to. Note, R ¹ is straight-chain alkyl group of C4, to the N to which R ² and R ³ are they are bound together with the hetero atom A compound which is a 6-membered azacycloalkyl group is diclonine There. Halogen is, F, Cl, I, Br, I is preferred.)
The pharmacologically acceptable salt is not limited as long as it forms a salt with those compounds, and specifically, for example, hydrochloride, sulfate, nitrate, hydrobromide, iodide Addition salts of inorganic acids such as hydrogen chloride, perchlorate and phosphate, oxalate, organic acid addition salts such as maleate, fumarate and succinate, methanesulfonate, ethane sulfone Acid salts, benzenesulfonic acid salts, p-toluenesulfonic acid salts, addition salts of sulfonic acids such as camphorsulfonic acid salts, addition salts of amino acids, etc., preferably hydrochlorides, oxalates, maleates , Methanesulfonate. Furthermore, it is needless to say that those compounds or their pharmacologically acceptable salts include not only anhydrate but also hydrates and crystalline polymorphs.

The glutathione concentration reducing agent is an agent that reduces glutathione concentration in cells. The glutathione concentration-lowering agent used in these antitumor agents is not limited, but preferred is an agent that inhibits the pathway in which glutathione is produced from cystine that is taken into the cell by xCT, such as xCT, Thioredoxin-1 (thioredoxin-1: TRX-1), glutamate-cysteine ligase (GCL) (EC 6.3.2.2) (also called γ-glutamylcysteine synthetase), or glutathione synthetase (EC 6.3.2.3) It is more preferred that the agent is an inhibitory agent, more preferably an xCT inhibitor. The xCT inhibitor is not particularly limited, but is preferably sulfasalazine, elastin, sorafenib, or an anti-xCT antibody.

A glutathione S-transferase inhibitor is an agent that inhibits the enzyme activity of glutathione S-transferase (EC 2.5. 1. 18), and in particular, HNE (4-HNE; 4-hydroxy-2-nonenal) -An agent that inhibits the activity of converting to GSH. The glutathione S-transferase inhibitors are not particularly limited, but glutathione analogs (eg, WO 95/08563, WO 96/40205, WO 99/54346, etc.), ketoprofen, indomethacin, ethacrynic acid, piroprost, anti-GST antibody, dominant negative mutant of GST Etc.

Here, "simultaneous administration" of two drugs means not only simultaneous administration in time but also in time as long as the other drug is administered while the effect of one drug remains. It also means to administer each alone. When two agents are simultaneously administered, two agents containing only one may be simultaneously administered, but two agents may be administered as one dosage form as a combination agent.

The administration target 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 a tumor containing a tumor cell resistant to a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor is preferable. The tumor cells may have high expression of aldehyde dehydrogenase. The glutathione concentration reducing agent or the glutathione S-transferase inhibitor is preferably an xCT inhibitor, more preferably sulfasalazine. Resistant tumor cells are tumor cells that survive in vivo when administered to patients usually at a therapeutic concentration for the usual treatment days, and have a 50% survival rate of 80% or more cell lines in vitro It refers to tumor cells with a survival rate of 90% or more at a concentration below. For example, a sulfasalazine-resistant tumor cell is a tumor cell that survives in vivo when administered for 2 weeks at 50-300 μg · h / mL of AUC _0-24 in vivo, and in vitro the survival rate is 200 μM. Refers to 90% or more of tumor cells. It is preferred that the sulfasalazine resistant tumor cells also have low levels of CD44v expression or be negative. With tumor cells in which aldehyde dehydrogenase is overexpressed, the gene expression of any of ALDH1A1, ALDH2, ALDH1B1 and ALDH3A1 is expressed at a level 3 times or more, preferably 10 times or more higher than that of OSC19 cells. Refers to cells that are The tumor to be treated may be contaminated with tumor cells expressing CD44v. This is because sulfasalazine has an anti-tumor effect effectively on tumor cells in which CD44v is expressed. Tumor cells expressing CD44v may be cells in which CD44v expression can be detected, but highly expressing cells are preferable. In this case, is high expression as high as the average level of ovarian tumor cells? Although it may be high, it is preferably 2 times or more, more preferably 4 times or more, and still more preferably 10 times or more.

The type of tumor is not particularly limited, but is preferably solid cancer, and is exemplified by colorectal adenocarcinoma, gastric adenocarcinoma, breast adenocarcinoma, lung adenocarcinoma, pancreatic adenocarcinoma, squamous cell carcinoma of the head and neck, ovarian tumor, testicular tumor be able to.

The antitumor agent may be formulated into tablets, powders, granules, powders, capsules, solutions, emulsions, suspensions and the like by conventional methods. At that time, it is manufactured using pharmaceutically acceptable additives known to those skilled in the art, such as excipients and carriers.

The antitumor agent may be administered in a manner suitable for an administration subject within the effective amount range. The effective amount can be appropriately determined finally by the judgment of a doctor or veterinarian in consideration of the type of dosage form, administration method, age and weight of administration subject, medical condition of administration subject, and the like. For example, the dose of the compound per day is preferably 0.1 mg / kg or more, more preferably 1 mg / kg or more, still more preferably 10 mg / kg or more, and 1000 mg / kg or less. Is preferably 300 mg / kg or less, more preferably 100 mg / kg or less. The administration method is not particularly limited. For example, it may be orally administered, may be parenterally administered by injection or infusion into the abdominal cavity or vein, or may be directly administered into cancer by injection etc. It is also good.

== Method of measuring growth rate or cell viability of tumor cells ==
One embodiment of the present invention comprises simultaneously administering an aldehyde dehydrogenase inhibitor, a glutathione concentration-reducing agent or a glutathione S-transferase inhibitor to tumor cells in vitro, and a growth rate of a tumor cell to which a drug has been administered. Or a step of measuring cell viability. The aldehyde dehydrogenase inhibitor, the glutathione concentration reducing agent, and the glutathione S-transferase inhibitor in this section are in accordance with those described in the "antitumor agent" section.

Since the aldehyde dehydrogenase inhibitor and the glutathione concentration reducing agent or the glutathione S-transferase inhibitor have a combined effect on the antitumor activity, a drug combination having a high combined effect can be found by this measurement method. Particularly effective tumor cells can be found for certain drug combinations.

Specifically, the growth rate or cell survival rate of a tumor cell to which a specific glutathione concentration reducing agent or a glutathione S-transferase inhibitor and a plurality of aldehyde dehydrogenase inhibitors were simultaneously administered to tumor cells in vitro and the drug was administered By measuring the rate, it is possible to identify an aldehyde dehydrogenase inhibitor that has a combined effect with a specific glutathione concentration reducing agent or glutathione S-transferase inhibitor. In addition, a specific aldehyde dehydrogenase inhibitor and multiple glutathione concentration reducing agents or glutathione S-transferase inhibitors are simultaneously administered to tumor cells in vitro to measure the growth rate or cell viability of the drug-administered tumor cells. By doing this, it is possible to identify a glutathione concentration reducing agent or a glutathione S-transferase inhibitor that has a combined effect with a specific aldehyde dehydrogenase inhibitor. Alternatively, a specific combination of an aldehyde dehydrogenase inhibitor and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor is simultaneously administered to a plurality of tumor cells in vitro, and the growth rate or cells of a plurality of tumor cells to which the agent is administered By measuring the survival rate, it is possible to identify a tumor cell that exerts a combined effect of an aldehyde dehydrogenase inhibitor and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor.

EXPERIMENTAL EXAMPLE 1 Combined Effect of Sulfasalazine and Diclonine (Purpose) In this example, the combined effect of sulfasalazine and diclonine having the xCT inhibitory effect is shown to reduce the survival rate of sulfasalazine resistant cells.

(Method) The oral squamous cell carcinoma cell line HSC-4, which is a sulfasalazine resistant cell line, was seeded at 2000 cells / well in a 96-well plate, and the culture was started. The medium used was DMEM. After 24 hours, the medium was changed to a medium containing 50 μM dicronine or an equivalent amount of DMSO and 0 μM (no addition), 50 μM, 100 μM, 200 μM, or 400 μM sulfasalazine, and the culture was continued for 48 hours. Thereafter, the cell viability was measured using Celltiter-Glo (Promega), and the cell viability of each case was calculated with the cell survival number of the control (with DMSO and without sulfasalazine added) being 100%. In FIG. 1, a graph showing the survival rate for each concentration of sulfasalazine was created.

(Results) HSC4 is a sulfasalazine resistant cell line, and sulfasalazine alone has little effect on cell viability. Furthermore, even with diclonine alone (with diclone and with no sulfasalazine added), the survival rate is 80%. However, when both diclonine and sulfasalazine are added, the survival rate becomes less than 10% when the amount of sulfasalazine is 100 μM or more.

Thus, sulfasalazine and diclonine have a combined effect on the reduction in the survival rate of sulfasalazine resistant cells.

(Experimental Example 2) Change in sensitivity to dyclonine by xCT knockdown (Objective) In this experimental example, even if knocking down xCT instead of sulfasalazine having xCT inhibitory effect, similar combined effect with dicronine can be obtained. By showing, it is shown that the combined effect of sulfasalazine and diclonine is mediated through the xCT inhibitory effect of sulfasalazine.

(Method) The oral squamous cell carcinoma cell line HSC-4 cells, a sulfasalazine-resistant cell line, are seeded at 3000 / well in a 96-well plate, and a non-silencing control (Scramble (Sense: UUCUCCGACGUGUCACGUtt (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: CCAGAACAUUACAAUAAAUtt (SEQ ID NO: 5) , Antisense: AUUAUUUGUAAUGUCUGGT (SEQ ID NO: 6)) was lipofected using Lipofectamine RNAiMAX (ThermoFisher Scientific) and culture was initiated. The medium used was DMEM. After 24 hours, the medium was changed to a medium containing 50 μM diclonine (solvent: DMSO) or an equivalent amount of DMSO, and the culture was continued for 48 hours. Thereafter, the cell viability was measured using Celltiter-Glo (Promega), and the cell viability of each control (non-silencing control, addition of DMSO) was calculated as 100%. The results are shown in FIG.

(Results) HSC-4 has about 60% cell viability with 50 μM diclone alone, but only about 10-20% cell viability with 50 μM diclone when knocked down xCT Do not have.

Thus, the combined effect of sulfasalazine and diclonine is through the xCT inhibitory effect of sulfasalazine.

(Experimental Example 3) Combined Effect of Sulfasalazine, Elastin, or BSO and Diclonine in Various Cancer Cell Lines (Objective) In this example, sulfasalazine, elastin, which is a specific inhibitor of xCT, or various tumor cells in various tumor cell lines As well as showing that BSO, which is a glutathione synthesis inhibitor, and dyclonine exert a combined effect, it also shows that xCT inhibition is mediated through glutathione synthesis inhibition.

(Method) In a 96-well plate, the cell line shown in FIG. 3 was seeded at 3000 cells / well and culture was started. The medium used was DMEM. After 24 hours, replace the medium with 50 μM dicronine or an equivalent amount of DMSO, 0 μM (no addition) or 400 μM sulfasalazine, 0 μM (no addition) or 5 μM elastin, 0 μM (no addition) or 100 μM BSO, The culture was continued for 48 hours. Thereafter, the cell viability was measured using Celltiter-Glo (Promega), and the cell viability of each of the controls (with DMSO added and without diclonine added) was calculated as 100%. The cell viability in each case is illustrated in FIG.

(Results) Although the size varies depending on the cell, the same combined effect was observed in any combination of sulfasalazine, elastin, or BSO and diclonine.

Thus, inhibition of xCT by sulfasalazine or elastin exerts its antitumor effect by inhibiting the synthesis of glutathione. Therefore, glutathione concentration reducing agents or glutathione S-transferase inhibitors can be used in place of sulfasalazine or elastin.

(Experimental Example 4) Combined Effect of Sulfasalazine and Diclonine in Vivo (Objective) In this example, the combined effect of sulfasalazine and diclonine is observed also in vivo.

(Method) 1 × 10 ⁶ cells of the oral squamous cell carcinoma cell line HSC-2 which is a sulfasalazine-resistant cell line are subcutaneously implanted in nude mice, and physiological saline, sulfasalazine alone, diclonine alone from the fourth day after transplantation Both sulfasalazine and dyclonine were administered intraperitoneally at a dose of sulfasalazine 400 mg / kg sulfasalazine 5 mg / kg once a day, and continued until day 22. The short diameter and the long diameter of the tumor were measured every 3 to 4 days, the tumor volume was calculated by the following equation, and the results were graphed in FIG.

Tumor volume = (major axis × (minor axis) ² ) / 2
Statistical analysis of tumor volume was performed on day 22 by t-test.

(Results) As shown in FIG. 4, each drug alone reduced the tumor volume by about 35%, but when both were administered, the volume was reduced by about 70%.

Thus, the combined administration of sulfasalazine and diclonine can reduce the growth of sulfasalazine resistant tumors.

(Experimental Example 5) Inhibition of ALDH by Diclonine (Objective) In this experimental example, it is shown that diclonine has an inhibitory activity of ALDH.

(Method) A 10 cm cell culture dish was inoculated with 8 × 10 ⁵ cells / cell of oral squamous cell carcinoma cell line HSC-4 cells / dish and culture was started. The medium used was DMEM. After 24 hours, the medium was replaced with a medium containing 50 μM diclonine (solvent: DMSO), and cultured for 24 hours. Thereafter, the cells were recovered, and cells having ALDH activity in the presence of N, N-diethylaminobenzaldehyde (DEAB) were stained with ALDEFLUOR kit (STEMCELL Technologies) and analyzed by FACS (in the figure, Dyclonine). As a control, the cells were not stained with ALDEFLUOR kit without addition of DEAB (Unstained in the figure), replaced with a medium containing an equivalent amount of DMSO without dyclonine and stained with ALDEFLUOR kit (Non-treatment in the figure) The experimental results for In addition, for measurement of positive cells, a gate is prepared so that positive cells become almost 0% for DMSO-treated samples (DEAB in the figure) stained with ALDEFLUOR kit in the presence of DEAB, and the positive rate in each case is Calculated.

(Results) As shown in FIG. 5, although approximately 25% of cell groups with high ALDH activity exist in DMSO-treated cells, ALDH activity is high in dyclonine-treated cells and in DEAB-treated cells that are known ALDH inhibitors. The high cell population is suppressed to about 1%.

Thus, diclonine has an inhibitory activity of ALDH.

(Experimental Example 6) Accumulation of HNE by Combination of Sulfasalazine and Diclonine (Purpose) In this example, the combination of sulfasalazine and diclonine significantly increases the level of HNE in tumor cells and the frequency of cells accumulating HNE. Indicates

(Method) HSC-4 cells are cultured in a medium containing 50 μM dicronine or an equivalent amount of DMSO and 0 μM (without addition) or 400 μM sulfasalazine in the same manner as in Experimental Example 1, and the treated cells are 4% PFA. -Fixed with PBS. Furthermore, after permeabilizing the cell membrane with 0.2% Triton X 100-PBS, blocking with 3% BSA-PBS was performed. Thereafter, fluorescent staining was performed using an anti-HNE antibody as a primary antibody and an Alexafluor 488-labeled anti-mouse IgG antibody as a secondary antibody. Similarly, antibody staining was performed using cells incubated with 50 μMHNE for 30 minutes as a positive control. An observation image with a fluorescence microscope is shown in FIG.

(Results) When diclonine or sulfasalazine was treated alone, an increase in intracellular HNE concentration was observed at a low frequency, but a combination of sulfasalazine and dyclonine resulted in a high frequency of accumulation of intracellular HNE. The

Thus, by combining the xCT inhibitor and the ALDH inhibitor, accumulation of intracellular HNE at high frequency and high concentration is observed. The reason for this is not limited to the following theory, but as shown in FIG. 11, there are a plurality of pathways that degrade HNE in cells, and among them, a GST-mediated pathway and an ALDH-mediated degradation pathway It is believed that HNE accumulates in cells by simultaneously inhibiting the two. And because HNE is cytotoxic, it is thought that tumor cells can not grow.

(Experimental example 7) Combined effect of sulfasalazine or BSO and diclonine analog (having a diclonine skeleton) (objective) The following diclonine analog (I) having a diclonine skeleton has a combined effect with sulfasalazine or BSO Show.

(Wherein, R ¹ is a C 1-6 linear or branched alkyl group, and R ² and R ³ are independently selected C 1-6 linear or branched alkyl groups, or R ² and R 3 ³ is taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group having the hetero atom N to which they are bonded, and R ⁴ is hydrogen or halogen. R ¹ is preferably a C 4 to 5 linear or branched alkyl group, and R ² and R ³ are C 2 alkyl groups, or R ² and R ^{3 taken} together are the N to which they are bonded is preferably a 6-membered azacycloalkyl group ring to. Note, R ¹ is straight-chain alkyl group of C4, to the N to which R ² and R ³ are they are bound together with the hetero atom A compound which is a 6-membered azacycloalkyl group is diclonine There. Halogen is, F, Cl, I, Br, I is preferred.)
(Method) Similar to Experimental Example 1, 0 μM (no addition), 25 μM, 50 μM, or 100 μM dyclonine, or 12.5 μM, 25 μM, 50 μM, 100 μM dyclonine analogue BAS00363846, STL327701, PHAR033081, PHAR298639, or Aldi- HSC-4 cells were cultured in a medium containing 2 (see FIG. 7B for the structural formula) and 0 μM (no addition) or 100 μMBSO or 300 μM sulfasalazine, and cell viability was measured and graphed in FIG. 7A.

(Results) All of these compounds exhibited combined effects with BSO or sulfasalazine.

Thus, the diclonine analogue (I) having a diclonine skeleton has a combined effect as an xCT inhibitor and an antitumor agent.

(Experimental Example 8) Combined Effect of BSO and Diclonine Analogue (Without a Diclonine Skeleton) (Objective) It is shown that a diclonine analogue without a diclonine skeleton does not have a combined effect with BSO.

(Method) Similar to Experimental Example 1, 0 μM (no addition), 12.5 μM, 25 μM, or 50 μM dyclonine, or 3.125 μM, 6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM dyclonine analogue (4 HSC-4 cells were cultured in a medium containing the structural formula (see FIG. 8B) and 0 μM (no addition) or 100 μMBSO, and the cell viability was measured and graphed in FIG. 8A.

(Results) The diclonine analogues not having a diclonine skeleton did not exhibit the combined effect with BSO.

Thus, the diclonine backbone is important for interaction with xCT inhibitors.

(Experimental Example 9) Combined Effect of Sulfasalazine, Elastin, or BSO and Diclonine in Sulfasalazine-Resistant OSC 19 Cells (Objective) Diclonine Has Combined Effect with a Glutathione Synthesis Inhibitor in Cancer Cell Lines That Acquired Resistance to xCT Inhibitor Indicates that.

(Method) 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. Parent strains of OSC19 cells or OSC19-SSZR cells are seeded at 3000 cells / well in a 96-well plate and cultured for 24 hours, after which the concentrations of sulfasalazine, elastin or BSO shown in FIG. 9 and 50 μM diclonine (solvent is DMSO) Alternatively, the medium was replaced with a medium containing an equal volume of DMSO and cultured for 48 hours. Thereafter, the cell viability was measured by Celltiter-Glo (Promega), and the cell viability was calculated with 100% of a control (sulfasalazine, elastin and BSO added, DMSO added) as 100%.

(Results) Diclonine also showed a combined effect with sulfasalazine, elastin or BSO in OSC19-SSZR cells.

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

(Experimental Example 10) Expression of ALDH Gene Family in Sulfasalazine-Resistant OSC 19 Cells and HSC-4 (Objective) It is shown that ALDH gene family is highly expressed in cancer cells having resistance to xCT inhibitor.

(Method) Messenger RNA was extracted from HSC-4 cells, OSC19 cells and OSC19-SSZR cells, and reverse transcription was performed to synthesize complementary DNA. Thereafter, using the obtained complementary DNA as a template, ALDHIAl, by quantitative RT-PCR.
The expression levels of ALDHIB1, 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 is graphed in FIG.

(Results) ALDHIAl was up-regulated in OSC19-SSZR compared to OSC19. ALDHIB1 and ALDH2 showed high expression in HSC-4. ALDH3Al was highly expressed in HSC4 and OSC19-SSZR. Thus, expression of ALDH family genes tended to be high in xCT hyposensitive cancer cell lines.

Thus, in cancer cells with high expression of ALDH family genes, HNE is degraded by ALDH family genes, so even if xCT inhibitor suppresses degradation to GST, HNE toxicity does not work , XCT inhibitor (see Figure 11). Administration of an ALDH inhibitor to such cells increases the sensitivity to the xCT inhibitor, and therefore, an antitumor agent containing an ALDH inhibitor and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor results in expression of an ALDH family gene. It works effectively on high cancer cells.

The present invention has made it possible to provide novel antitumor agents and combination agents.

Claims

An antitumor agent, which comprises a glutathione concentration reducing agent or a glutathione S-transferase inhibitor as an active ingredient, which is administered simultaneously with an effective amount of an aldehyde dehydrogenase inhibitor.
An antitumor agent which comprises an aldehyde dehydrogenase inhibitor as an active ingredient, which is administered simultaneously with an effective amount of a glutathione concentration reducing agent or a glutathione S-transferase inhibitor.
The above-mentioned glutathione concentration-lowering agent is xCT, Thioredoxin-1 (thioredoxin-1: TRX-1), glutamate-cysteine ligase (GCL) (EC 6.3.2.2) (also called γ-glutamylcysteine synthetase), glutathione synthesis The antitumor agent according to claim 1 or 2, which is an agent that inhibits any activity of an enzyme (EC 6.3.2.3).
The antitumor agent according to claim 3, wherein the agent is an inhibitor of xCT transporter.
The antitumor agent according to claim 4, wherein the inhibitor of the xCT transporter is sulfasalazine, elastin or sorafenib.
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.

(Wherein, R 1 is a C 1-6 linear or branched alkyl group, and R 2 and R 3 are independently selected C 1-6 linear or branched alkyl groups, or R 2 and R 3 3 is taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group having the hetero atom N to which they are bonded, and R 4 is hydrogen or halogen. )
The antitumor agent according to claim 6, wherein the compound represented by the formula (I) is dyclonine, BAS00363846, STL327701, PHAR033081, PHAR298639, or Alid-2.
A combination drug comprising an aldehyde dehydrogenase inhibitor and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor as active ingredients.
The combination drug according to claim 8, wherein the glutathione concentration reducing agent is an inhibitor of xCT transporter.
The combination drug according to claim 9, wherein the inhibitor of the xCT transporter is sulfasalazine, elastin or sorafenib.
The combination drug according to any one of claims 8 to 10, wherein the aldehyde dehydrogenase inhibitor is a compound represented by the formula (I) or a pharmacologically acceptable salt thereof.

(Wherein, R 1 is a C 1-6 linear or branched alkyl group, and R 2 and R 3 are independently selected C 1-6 linear or branched alkyl groups, or R 2 and R 3 3 is taken together to form a 4-, 5-, 6-, or 7-membered azacycloalkyl group having the hetero atom N to which they are bonded, and R 4 is hydrogen or halogen. )
The combination drug according to claim 11, wherein the compound represented by the formula (3) is diclonine, BAS00363846, STL327701, PHAR033081, PHAR298639, or Alid-2.
An antitumor agent comprising the combination according to any one of claims 8 to 12.
The antitumor agent according to any one of claims 1 to 7, which is an antitumor agent for a tumor comprising a tumor cell resistant to a glutathione concentration-lowering agent or a glutathione S-transferase inhibitor.
The antitumor agent according to claim 14, wherein the glutathione concentration reducing agent is an xCT inhibitor.
The antitumor agent according to claim 15, wherein the xCT inhibitor is sulfasalazine.
The antitumor agent according to any one of claims 14 to 16, wherein aldehyde dehydrogenase is highly expressed in the tumor cells.
The antitumor agent according to any one of claims 14 to 17, wherein the tumor further comprises a tumor cell expressing CD44v.
Simultaneously administering an aldehyde dehydrogenase inhibitor and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor to tumor cells in vitro;
Measuring the growth rate or cell viability of the tumor cells.
A method for identifying an aldehyde dehydrogenase inhibitor that has a combined effect with a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, comprising
Simultaneously administering a specific glutathione concentration reducing agent or glutathione S-transferase inhibitor and a plurality of aldehyde dehydrogenase inhibitors to tumor cells in vitro;
Measuring the growth rate or cell viability of the tumor cells.
A method for identifying a glutathione concentration reducing agent or a glutathione-S-transferase inhibitor which has an effect in combination with an aldehyde dehydrogenase inhibitor, comprising
Simultaneously administering a specific aldehyde dehydrogenase inhibitor, which is an antitumor agent, and a plurality of glutathione concentration reducing agents or glutathione S-transferase inhibitors to tumor cells in vitro;
Measuring the growth rate or cell viability of the tumor cells.
A method for identifying a tumor cell that exerts a combined effect of an aldehyde dehydrogenase inhibitor and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor, comprising
Simultaneously administering a specific combination of an aldehyde dehydrogenase inhibitor and a glutathione concentration reducing agent or a glutathione S-transferase inhibitor to a plurality of tumor cells in vitro;
Measuring the growth rate or cell viability of the plurality of tumor cells.
The measurement method according to claim 19, wherein the tumor cell is resistant to a glutathione concentration reducing agent or a glutathione S-transferase inhibitor.
The specific method according to any one of claims 20 to 22, wherein the tumor cell is resistant to a glutathione concentration reducing agent or a glutathione S-transferase inhibitor.