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WO2019098288A1 - Agent antitumoral et agent de formulation - Google Patents

Agent antitumoral et agent de formulation Download PDF

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Publication number
WO2019098288A1
WO2019098288A1 PCT/JP2018/042327 JP2018042327W WO2019098288A1 WO 2019098288 A1 WO2019098288 A1 WO 2019098288A1 JP 2018042327 W JP2018042327 W JP 2018042327W WO 2019098288 A1 WO2019098288 A1 WO 2019098288A1
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glutathione
inhibitor
aldehyde dehydrogenase
agent
reducing agent
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PCT/JP2018/042327
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English (en)
Japanese (ja)
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永野　修
秀行 佐谷
章悟 岡崎
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学校法人 慶應義塾
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Priority to JP2019554284A priority Critical patent/JP7388702B2/ja
Publication of WO2019098288A1 publication Critical patent/WO2019098288A1/fr
Priority to JP2023191878A priority patent/JP2024023269A/ja

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/12—Ketones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/13—Amines
    • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
    • A61K31/137—Arylalkylamines, e.g. amphetamine, epinephrine, salbutamol, ephedrine or methadone
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
    • A61K31/445—Non condensed piperidines, e.g. piperocaine
    • A61K31/4453—Non condensed piperidines, e.g. piperocaine only substituted in position 1, e.g. propipocaine, diperodon
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/63—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide
    • A61K31/635—Compounds containing para-N-benzenesulfonyl-N-groups, e.g. sulfanilamide, p-nitrobenzenesulfonyl hydrazide having a heterocyclic ring, e.g. sulfadiazine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/39—Connective tissue peptides, e.g. collagen, elastin, laminin, fibronectin, vitronectin, cold insoluble globulin [CIG]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/15—Medicinal preparations ; Physical properties thereof, e.g. dissolubility
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing

Definitions

  • the present invention relates to antineoplastic agents and combinations.
  • 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.
  • 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.
  • GSH glutathione
  • 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.
  • 5-ASA is the main active ingredient for the above-mentioned diseases.
  • 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.
  • An object of the present invention is to provide novel antitumor agents and combination agents.
  • 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.
  • 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 ⁇ -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.
  • R 1 is a C 1-6 linear or branched alkyl group
  • 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
  • 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.
  • the tumor cell may be resistant to a glutathione concentration-reducing agent or a glutathione S-transferase inhibitor.
  • 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.
  • HNE 4-HNE; 4-hydroxy-2-nonenal
  • 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.
  • it is the graph which showed expression of the ALDH gene family in HSC4 cell, OSC19 cell, or sulfasalazine resistant OSC19 cell.
  • HNE 4-hydroxy-2-nonenoic acid
  • GSH glutathione
  • ALDH aldehyde dehydrogenase
  • GST glutathione S-transferase
  • 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.
  • 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.
  • 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).
  • ALDH aldehyde dehydrogenase 2
  • 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 salt
  • R 1 is a C 1-6 linear or branched alkyl group
  • 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
  • R 4 is hydrogen or halogen.
  • R 1 is preferably a C 4 to 5 linear or branched alkyl group
  • 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.
  • 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,
  • 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 ⁇ -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.
  • spontaneous 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.
  • 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.
  • a sulfasalazine-resistant tumor cell is a tumor cell that survives in vivo when administered for 2 weeks at 50-300 ⁇ g ⁇ h / mL of AUC 0-24 in vivo, and in vitro the survival rate is 200 ⁇ M. 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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)
  • the combined effect of sulfasalazine and diclonine having the xCT inhibitory effect is shown to reduce the survival rate of sulfasalazine resistant cells.
  • 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 ⁇ M dicronine or an equivalent amount of DMSO and 0 ⁇ M (no addition), 50 ⁇ M, 100 ⁇ M, 200 ⁇ M, or 400 ⁇ M sulfasalazine, and the culture was continued for 48 hours.
  • FIG. 1 a graph showing the survival rate for each concentration of sulfasalazine was created.
  • 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 ⁇ M or more.
  • sulfasalazine and diclonine have a combined effect on the reduction in the survival rate of sulfasalazine resistant cells.
  • 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
  • the medium used was DMEM. After 24 hours, the medium was changed to a medium containing 50 ⁇ M 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.
  • HSC-4 has about 60% cell viability with 50 ⁇ M diclone alone, but only about 10-20% cell viability with 50 ⁇ M diclone when knocked down xCT Do not have.
  • 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 ⁇ M dicronine or an equivalent amount of DMSO, 0 ⁇ M (no addition) or 400 ⁇ M sulfasalazine, 0 ⁇ M (no addition) or 5 ⁇ M elastin, 0 ⁇ M (no addition) or 100 ⁇ M BSO, The culture was continued for 48 hours.
  • glutathione concentration reducing agents or glutathione S-transferase inhibitors can be used in place of sulfasalazine or elastin.
  • Tumor volume (major axis ⁇ (minor axis) 2 ) / 2 Statistical analysis of tumor volume was performed on day 22 by t-test.
  • the combined administration of sulfasalazine and diclonine can reduce the growth of sulfasalazine resistant tumors.
  • 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 ⁇ M 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).
  • 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)
  • diclonine has an inhibitory activity of ALDH.
  • HSC-4 cells are cultured in a medium containing 50 ⁇ M dicronine or an equivalent amount of DMSO and 0 ⁇ M (without addition) or 400 ⁇ M 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 ⁇ MHNE for 30 minutes as a positive control. An observation image with a fluorescence microscope is shown in FIG.
  • HNE intracellular HNE at high frequency and high concentration.
  • 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.
  • R 1 is a C 1-6 linear or branched alkyl group
  • 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
  • R 4 is hydrogen or halogen.
  • R 1 is preferably a C 4 to 5 linear or branched alkyl group
  • 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.
  • 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.
  • 25 ⁇ M, 50 ⁇ M, or 100 ⁇ M dyclonine, or 12.5 ⁇ M, 25 ⁇ M, 50 ⁇ M, 100 ⁇ M 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 ⁇ M (no addition) or 100 ⁇ MBSO or 300 ⁇ M sulfasalazine, and cell viability was measured and graphed in FIG. 7A.
  • the diclonine analogue (I) having a diclonine skeleton has a combined effect as an xCT inhibitor and an antitumor agent.
  • the diclonine backbone is important for interaction with xCT inhibitors.
  • 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 ⁇ M diclonine (solvent is DMSO)
  • the medium was replaced with a medium containing an equal volume of DMSO and cultured for 48 hours.
  • 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%.
  • dyclonine shows a combined effect with a glutathione synthesis inhibitor even in cancer cells that have acquired resistance to xCT inhibitors.
  • 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 ⁇ Ct method, and is graphed in FIG.
  • 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.
  • expression of ALDH family genes tended to be high in xCT hyposensitive cancer cell lines.
  • 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.

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Abstract

L'invention concerne : un nouvel agent antitumoral qui contient, en tant que principe actif, un agent abaissant la concentration de glutathion ou un inhibiteur de la glutathion S-transférase qui est administré simultanément avec une quantité efficace d'un inhibiteur de l'aldéhyde déshydrogénase ; ou un nouvel agent antitumoral qui contient, en tant que principe actif, un inhibiteur de l'aldéhyde déshydrogénase qui est administré simultanément avec une quantité efficace d'un agent abaissant la concentration de glutathion ou d'un inhibiteur de la glutathion S-transférase ; ou un nouvel agent de formulation qui contient, en tant que principes actifs, un inhibiteur de l'aldéhyde déshydrogénase et un agent abaissant la concentration de glutathion ou un inhibiteur de la glutathion S-transférase.
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CN114126652A (zh) * 2019-05-14 2022-03-01 学校法人庆应义塾 抗肿瘤剂和配合剂

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