JP6993666B2 - Anti-cancer agent - Google Patents

Description

The present invention relates to an anticancer agent using a low molecular weight compound, particularly an anticancer agent effective for early stage lung adenocarcinoma.

Currently, lung cancer is the most common cause of cancer death in Japan, and its 5-year survival rate is extremely low at 40% or less. Among them, lung adenocarcinoma has the highest incidence and has been increasing in recent years. In advanced adenocarcinoma, molecular targeted therapy for abnormalities such as EGFR mutation and ELM4-ALK fusion has come to be performed, and gefitinib for EGFR mutant lung adenocarcinoma and crizotinib for lung adenocarcinoma with EML4-ALK fusion etc. It has been known.
However, companion diagnostics are essential because existing drugs must have the targeted gene mutations, which is an absolute requirement for therapeutic indication. Also, all of these treatments are for advanced lung adenocarcinoma, and there is no cure for early stage lung adenocarcinoma.
Stratifin (SFN) is an anchor protein belonging to the 14-3-3 family, and is a tumor malignant factor whose expression is upregulated in almost all lung adenocarcinoma cases (Non-Patent Documents 1 and 2). Therefore, compounds that can suppress / delay the upregulation of SFN and compounds that can inhibit its function are used for all lung adenocarcinomas and various cancers including lung adenocarcinoma without companion diagnostics. Can be expected to show an effect.
We have identified SKP1 as a binding factor for SFN. SKP1 is an adapter portion of SCF, which is an E3 ubiquitin ligase, and SFN is thought to cause malignant transformation of cancer by binding to the SKP1 protein. Therefore, a compound capable of inhibiting the binding between SFN and SKP1 is promising as an anticancer agent.
On the other hand, USP8, which is one of the deubiquitinating enzymes, has a binding motif to the 14-3-3 protein, and deubiquitins the EGF receptor activated by the epidermal growth factor EGF by binding to SFN. It is known that the transport / degradation to lysosomes is negatively regulated by the conversion (Non-Patent Document 3).

Shiba-Ishii A, et al., Int J Cancer 129, 2445-2453 (2011) Shiba-Ishii A, et al., Molecular Cancer 14: 142 (2015) Noguchi M, et al., IASLC2015 (the 16th IASLC World Conference on Lung Cancer) P1.04-071 (2015)

The present invention has found a compound capable of suppressing / delaying the upregulation of SFN and a compound capable of inhibiting the function thereof, and provides an anticancer agent effective for cancer in general, particularly early stage cancer, using the compound. The purpose is to do.

In order to solve the above-mentioned problems, the present inventors have described (i) SFN and (ii) SKP1 and USP8 as compounds capable of suppressing / delaying the upregulation of SFN in cancer and compounds capable of inhibiting the function thereof. Screening was performed using the suppression of binding to at least one as an index, multiple identifications were made from the existing drug library, and it was confirmed that these compounds actually suppress the growth of multiple cancer cells including lung adenocarcinoma. The invention was completed.
That is, the present invention is as follows.
[1] An anticancer agent containing, as an active ingredient, a compound that inhibits the binding of (i) Stratifin and (ii) at least one of SKP1 and USP8.
[2] The anticancer agent according to the above [1], wherein the compound is a compound represented by the following formula or an analog thereof.

[3] Cancers are breast cancer, prostate cancer, pancreatic cancer, stomach cancer, lung cancer, colon cancer, esophageal cancer, duodenal cancer, head and neck cancer, brain tumor, neurothatomy, non-small cell lung cancer, lung. Small cell cancer, liver cancer, kidney cancer, bile duct cancer, uterine cancer, ovarian cancer, bladder cancer, skin cancer, hemangiomas, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, Hemangiomas, hemangiofibroma, retinal sarcoma, penis cancer, pediatric solid cancer, Kaposi sarcoma, Kaposi sarcoma caused by AIDS, maxillary sinus tumor, fibrous histiocytoma, smooth myoma, rhizome myoma, and leukemia The anticancer agent according to the above [1] or [2], which is at least one selected from.
[4] The anticancer agent according to any one of the above [1] to [3], wherein the cancer is early stage lung adenocarcinoma.

According to the present invention, cancer can be effectively treated without the need for companion diagnostics. In addition, in the case of lung adenocarcinoma, since SFN is upregulated from the stage of early invasive lung adenocarcinoma, treatment methods other than surgical excision have not been established so far for early stage lung adenocarcinoma. Effective chemical treatment can be provided.

6 is a Western blot image showing the results of examining the binding inhibitory ability of aprepitant to the binding between SFN and SKP1 or the binding between SFN and USP8. It is a graph which shows the result of having investigated the cell proliferation inhibitory ability of aprepitant.

Hereinafter, the present invention will be described. The terms used herein have meanings commonly used in the art unless otherwise noted.

The anticancer agent of the present invention contains, as an active ingredient, a compound capable of suppressing / delaying the upregulation of SFN expression, or a compound capable of inhibiting the function thereof. The compound is not particularly limited as long as it is a compound that suppresses the binding of (i) SFN to at least one of (ii) SKP1 and USP8 and has a cell proliferation inhibitory effect, but is specifically described by the following formula. Examples thereof include the compound represented by (1) or an analog thereof.

Aprepitant and its analogs <br />

The compound represented by is commonly known as aprepitant and is commercially available as an antiemetic agent. Examples of the analogs of the compound include the following compounds disclosed in WO95 / 16679 and pharmaceutically acceptable salts thereof.

(Definition of each symbol in the formula is as described in WO95 / 16679)
Preferably,
R ² and R ³ are independently hydrogen atoms, lower alkyl groups (eg, C _1-6 alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.) ), Lower alkenyl (eg, C _2-6 alkenyl group such as vinyl, allyl, isopropenyl, butenyl, isobutenyl, etc.) and phenyl;
R ⁶ , R ⁷ and R ⁸ are independently selected from the group consisting of hydrogen atom, lower alkyl group (synonymous with above), halogen (eg fluoro, chloro, bromo and iodine) and -CF ₃ ;
R ¹¹ , R ¹² and R ¹³ are independently selected from the group consisting of a hydrogen atom, a lower alkyl group (synonymous with the above), a halogen (eg, fluoro, chloro, bromo and iodine, preferably fluoro) and -CF ₃ . ;
Y is -O-;
Z is C _1-4 alkyl (eg, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, etc., preferably methyl);
R ¹ is a compound and a pharmaceutically acceptable salt thereof selected from the following formula.

Ticagrelor and its analogs <br />

The compound represented by is commonly known as ticagrelor and is commercially available as an antiplatelet agent. Examples of the analogs of the compound include the following compounds disclosed in WO99 / 05143 and pharmaceutically acceptable salts thereof.

(Definition of each symbol in the formula is as described in WO99 / 05143)
Preferably,
R ¹ may be substituted with trifluoromethyl, respectively, with C _1-4 alkyl (eg, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, etc., preferably propyl) or phenyl. can be;
R ² is butyl or cyclopropyl substituted with phenyl, the phenyl group itself being one or more (preferably two) halogens (preferably fluoro), C _3-8 alkyl (eg, methyl, ethyl, etc.). Propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.), alkoxy (eg, for example, methoxy, ethoxy, propoxy, isopropoxy, butoxy, isobutoxy, sec-butoxy, tert-butoxy, etc.) C _1-6 alkoxy group, etc.), substituted with phenoxy or phenyl;
Both R ³ and R ⁴ are hydroxy;
Compounds in which R is OH, CH ₂ OH, CH ₂ CH ₂ OH, OCH ₂ CH ₂ OH, CH ₂ OCH ₂ C (CH ₃ ) ₂ OH or OCH ₂ C (CH ₃ ) ₂ OH and pharmaceutically acceptable thereof. It is salt.

ZM241385 and its analogs <br />

The compound represented by (CAS No. 139180-30-6) is known as an adenosine receptor antagonist and is also sold as a reagent. Examples of the analog of the compound include the following compounds disclosed in JP-A-5-97855 and pharmaceutically acceptable salts thereof.

(Definition of each symbol in the formula is as described in Japanese Patent Application Laid-Open No. 5-97855)
Preferably,
Q is frill; R ¹ is hydrogen or acetyl; R ² is cyclopentyl, cyclohexyl, tetrafluorophenyl, pentafluorophenyl, pyridyl, thiadiazolyl, _C4 to C6 alkyl (eg, butyl, isobutyl, sec _- butyl). , Turt-butyl, etc.), optionally substituted phenyl (C ₁ to C ₂ ) alkyl (eg, phenylmethyl, phenylethyl, preferably phenylethyl), optionally substituted phenyl, frillmethyl or pyridylmethyl, said Either optionally substituted phenyl is unsubstituted or by methylenedioxy or fluoro, chloro, cyano, trifluoromethyl, methoxycarbonyl, hydroxy, pivaloyloxy, nitro, methyl, methoxy, t-butoxycarbonylethyl and sulfamoyl. Phenyl substituted by the above; X is oxy or imino; A is N or CT (T is hydrogen) and a pharmaceutically acceptable salt thereof.
Especially preferably
R ² is cyclohexyl, tetrafluorophenyl, 2-methylpropyl, phenyl, 2-fluorophenyl, 3-fluorophenyl, 2-chlorophenyl, 3-chlorophenyl, 2-cyanophenyl, 3-cyanophenyl, 2-nitrophenyl, 2 -Methenylcarbonylphenyl, 2-methoxyphenyl, 3-methoxyphenyl, 2-methylphenyl, 3-methylphenyl, 3-trifluoromethylphenyl, benzyl, 2-fluorobenzyl, 3-methoxybenzyl, 2-furylmethyl, 2 -Phenylethyl, 2- (4-chlorophenyl) ethyl, 2- (2-methylphenyl) ethyl, 2- (4-t-butoxycarbonylphenyl) ethyl, 2- (4-hydroxyphenyl) ethyl, 2- (4) -A compound that is sulfamoylphenyl) ethyl and 2- (4-pivaloyloxyphenyl) ethyl or a pharmaceutically acceptable salt thereof.

Ezetimibe and its analogs <br />

The compound represented by is commonly known as ezetimibe and is commercially available as a therapeutic agent for high cholesterol. Examples of the analogs of the compound include the following compounds disclosed in JP-A-8-509989 and pharmaceutically acceptable salts thereof.

(Definition of each symbol in the formula is as described in Japanese Patent Publication No. 8-509989)
Preferably,
X, Y and Z are identical and -CH _2- ;
R ¹ and R ³ are the same and are hydrogen atoms;
R and R ² are independently metabolized to -OR ⁶ or easily hydroxy (eg, -O (CO) R ⁶ , -O (CO) OR ⁹ and -O (CO) NR ⁶ R ⁷ can be;
R ⁶ , R ⁷ and R ⁸ are hydrogen, lower alkyl (eg, C _1-6 alkyl groups such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, hexyl, etc.), Selected independently from the group consisting of aryl (eg, _C6-10 aryl groups such as phenyl, naphthyl, etc.) and aryl (synonymous with above) substituted lower alkyl (synonymous with above);
R ⁹ is a lower alkyl (synonymous with above), aryl (synonymous with above) or aryl substituted lower alkyl (synonymous with above);
The sum of m, n, p, q and r is preferably 2, 3 or 4, more preferably 3 (more preferably m, n and r are 0, q is 1 and p is p. 2 or p, q and n are 0 respectively, r is 1 and m is 2 or 3)
A compound or a pharmaceutically acceptable salt thereof.
A compound in which m, n and r are 0, q is 1, p is 2, Z is _−CH2- , and R is −OR ⁶ (synonymous with the above, preferably a hydrogen atom). Alternatively, a pharmaceutically acceptable salt thereof is also preferable.
p, q and n are 0 respectively, r is 1, m is 2, X is -CH _2- , and R ² is -OR ⁶ (synonymous with the above, preferably a hydrogen atom). Compounds or pharmaceutically acceptable salts thereof are also preferred.

Chlorhexidine and its analogs <br />

The compound represented by is commonly called chlorhexidine and is commercially available as a bactericidal agent and a disinfectant. Examples of the analog of the compound include the following bis-diguanide compounds disclosed in JP-A-49-66838 and pharmaceutically acceptable salts thereof.

(Definition of each symbol in the formula is as described in JP-A-49-66838)
Preferably,
A and A'are p-chlorophenyl groups, respectively;
z and z'are 0;
A compound in which n is 6 and a pharmaceutically acceptable salt thereof.

In the present specification, pharmaceutically acceptable salts include, for example, trifluoroacetic acid, acetic acid, lactic acid, succinic acid, maleic acid, tartrate acid, citric acid, gluconic acid, ascorbic acid, benzoic acid, methanesulfonic acid, p-. Acid addition salts with acids such as toluenesulfonic acid, silicic acid, fumaric acid, phosphonic acid, hydrochloric acid, nitrate, hydrobromic acid, hydroiodic acid, sulfamic acid, sulfuric acid; for example, sodium, potassium, magnesium, calcium. Metal salts with the like; for example, salts with organic bases such as trimethylamine, triethylamine, pyridine, picolin, N-methylpyrrolidin, N-methylpiperidine, N-methylmorpholin and the like.

A compound that inhibits the binding of (i) SFN exemplified by the above compound used as an active ingredient in the present invention to at least one of (ii) SKP1 and USP8 is also referred to as the compound of the present invention in the present specification.

The compound of the present invention may be commercially available, or may utilize features based on the type of basic skeleton or substituent thereof, to which various known (previously reported) synthetic methods may be applied, and if desired. It can be appropriately modified and manufactured. For example, aprepitant and its analogs are described in WO95 / 16679, ticagrelor and its analogs are described in WO99 / 05143, and ZM241385 and its analogs are described in JP-A-5-97855. , Ezetimibe and its analogs by the method described in JP-A-8-509989, and chlorhexidine in Japanese Patent Application Laid-Open No. 49-66838 and US Pat. Nos. 2,684,924, 2,990,425, 2,830,006. No. and No. 2,863,019 and can be prepared according to these.

The compound of the present invention has isomers such as optical isomers, steric isomers, positional isomers, and rotational isomers, and either one isomer or a mixture of isomers can be used in the present invention. Each of these isomers can be obtained as a single product by a synthesis method and a separation method (concentration, solvent extraction, column chromatography, recrystallization, etc.) known per se.

The compound of the present invention may be crystalline or amorphous. When the phenyl hydroxide is crystalline, it is included in the phenyl hydroxide whether it has a single crystalline form or a crystalline mixture. Crystals can be produced by crystallization by applying a crystallization method known per se.

The compound of the present invention may be a solvate (for example, a hydrate or the like) or a non-solvate, and both are included in the phenyl hydroxide of the present invention.

The compound of the present invention may be labeled with an isotope (eg, ^3H , ^14C , ^35S , ^125I , etc.) or the like.

The compound of the present invention has a selective growth inhibitory effect on cancer cells, and is used in mammals (eg, humans, monkeys, cats, pigs, horses, cows, mice, rats, guinea pigs, dogs, rabbits, etc.). It is preferably useful as a preventive and / or therapeutic agent for cancer in humans. Here, "selective" means that although it exhibits a growth inhibitory effect on cancer cells, it is not shown on normal cells, or even if it is shown, the degree is significantly low.

Cancer cells are cells that are derived from somatic cells and have acquired infinite proliferative capacity. Cancers from which cancer cells are derived include, for example, lung cancer (for example, non-small cell lung cancer, small cell lung cancer, malignant mesotheloma, etc.) and breast cancer (for example, invasive duct cancer, non-invasive duct). Cancer, inflammatory breast cancer, etc.), prostate cancer (eg, hormone-dependent prostate cancer, hormone-independent prostate cancer, etc.), pancreatic cancer (eg, pancreatic duct cancer, etc.), gastric cancer (eg, papillary gland) Cancer, mucinous adenocarcinoma, glandular squamous epithelial cancer, etc.), colon cancer (eg, gastrointestinal stromal tumor, etc.), rectal cancer (eg, gastrointestinal stromal tumor, etc.), colon cancer (eg, gastrointestinal stromal tumor, etc.), colon cancer (eg, gastrointestinal stromal tumor, etc.) Familial colon cancer, hereditary non-polyposis colon cancer, gastrointestinal stromal tumor, etc.), esophageal cancer, duodenal cancer, tongue cancer, pharyngeal cancer (eg, nasopharyngeal cancer, mesopharyngeal cancer, etc.) Hypopharyngeal cancer, etc.), head and neck cancer, salivary adenocarcinoma, brain tumor (eg, pineapple stellate cell tumor, hairy cell stellate tumor, diffuse stellate tumor, degenerative stellate cell tumor, etc.), Liver cancer (eg, primary liver cancer, extrahepatic bile duct cancer, etc.), kidney cancer (eg, renal cell cancer, transitional epithelial cancer between renal pelvis and urinary tract, etc.), bile sac cancer, bile duct cancer , Pancreatic cancer, Endometrial cancer, Cervical cancer, Ovarian cancer (eg, epithelial ovarian cancer, extragonal embryonic cell tumor, ovarian embryonic cell tumor, low-grade ovarian tumor, etc.), bladder Hmm, urinary tract cancer, skin cancer (eg, intraocular (eye) melanoma, Mercel cell cancer, etc.), hemangiomas, malignant lymphoma (eg, non-Hodgkin lymphoma, Hodgkin's disease, etc.), melanoma (malignant melanoma) , Thyroid cancer (eg, thyroid medullary cancer, etc.), parathyroid cancer, nasal cavity cancer, sinus cancer, penis cancer, testicular tumor, pediatric solid cancer (eg, Wilms tumor, pediatric kidney tumor, etc.) ), Maxillous sinus tumor, etc., but not limited to these.
Preferably, the cancers are lung cancer, breast cancer, prostate cancer, pancreatic cancer, stomach cancer, colon cancer, esophageal cancer, duodenal cancer, head and neck cancer, brain cancer, liver cancer, kidney cancer, bile duct. At least selected from cancer, uterine cancer, ovarian cancer, bladder cancer, skin cancer, malignant lymphoma, malignant melanoma, thyroid cancer, bone tumor, penis cancer, pediatric solid cancer, maxillary sinus tumor It is one kind.
More preferably, the cancer is lung adenocarcinoma, breast cancer or colorectal cancer.
The growth inhibitory effect of cancer cells can be measured in vitro and / or in vivo according to a method usually practiced in the art or by modifying the method as necessary. For example, it can be measured by the method described in Examples described later.

The content of the compound of the present invention in the anticancer agent containing the compound of the present invention as an active ingredient is usually about 0.01 to about 99.9% by weight, preferably about 0.1 to about 50, based on the whole preparation. It is% by weight.

The dose of the compound of the present invention varies depending on the compound used, but if it is a compound already on the market, it is appropriately set based on the clinical dose thereof, and the age, body weight, general health condition, sex, diet, etc. It depends on the time of administration, method of administration, rate of excretion, combination of drugs, degree of medical condition being treated at the time of the patient, and other factors are taken into consideration.
For example, the daily dose of the compound of the present invention is about 0.01 to 10 mg / Kg (body weight), preferably about 0.5 to 5 mg / Kg (body weight), and more preferably about 0.1 to 1 mg / Kg (body weight). It is preferable to administer the body weight) in one dose or in two to three divided doses.

The compound of the present invention can be used in combination with other drugs depending on the target disease. These concomitant agents may be small molecule compounds, high molecular weight proteins, polypeptides, antibodies, vaccines and the like. In this case, the administration time of the compound of the present invention and the concomitant drug is not limited, and the compound of the present invention and the concomitant drug may be combined at the time of administration. For example, it can be used in combination with other anticancer agents that exhibit anticancer activity by a mechanism of action different from that of the compound of the present invention, such as cisplatin, gemcitabine, carboplatin, taxol, taxotere, methotrexate, vinblastine, and adriamycin.

The compound of the present invention may be blended with a pharmaceutically acceptable carrier to form a solid preparation such as a tablet, a capsule, a granule or a powder; a liquid preparation such as a syrup or an injection; a patch, an ointment, a plaster or the like. It can be appropriately formulated as a transdermal absorbent; an inhalant; a suppository.
The drug containing the compound of the present invention is orally or parenterally administered, and the above-mentioned compounds may be used alone or in combination of two or more.

As the pharmaceutically acceptable carrier, various organic or inorganic carrier substances commonly used as pharmaceutical material can be used. Specifically, it contains excipients, lubricants, binders, disintegrants, solvents, lysis aids, suspending agents, tonicity agents, buffers, pain-relieving agents, etc. in solid formulations. can do. Further, if necessary, pharmaceutical additives such as preservatives, antioxidants, colorants, and sweeteners can be used.

Examples of excipients include lactose, sucrose, glucose, starch, sucrose, microcrystalline cellulose, citrus powder, mannitol, sodium hydrogencarbonate, calcium phosphate, calcium sulfate and the like.

Examples of lubricants include magnesium stearate, stearic acid, calcium stearate, purified talc, colloidal silica and the like.

Examples of the binder include crystalline cellulose, sucrose, mannitol, dextrin, hydroxypropyl cellulose, hydroxypropylmethyl cellulose, polyvinylpyrrolidone and the like.

Examples of the disintegrant include starch, carboxymethyl cellulose, calcium carboxymethyl cellulose, sodium croscarmellose, sodium carboxymethyl starch and the like.

Preferable examples of the solvent include, for example, water for injection, alcohol, propylene glycol, macrogol, sesame oil, corn oil and the like.

Preferable examples of the solubilizing agent include polyethylene glycol, propylene glycol, D-mannitol, benzyl benzoate, ethanol, trisaminomethane, cholesterol, triethanolamine, sodium carbonate, sodium citrate and the like.

Examples of suspending agents include surfactants such as stearyltriethanolamine, sodium lauryl sulfate, laurylaminopropionic acid, lecithin, benzalkonium chloride, benzethonium chloride, glycerin monostearate; polyvinyl alcohol, polyvinylpyrrolidone, etc. Examples thereof include sodium carboxymethyl cellulose, methyl cellulose, hydroxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose and the like.

Preferable examples of the tonicity agent include, for example, sodium chloride, glycerin, D-mannitol and the like.

Suitable examples of the buffer include, for example, buffers such as phosphates, acetates, carbonates and citrates.
Preferable examples of the soothing agent include benzyl alcohol and the like.

Suitable examples of preservatives include, for example, paraoxybenzoic acid esters, chlorobutanol, benzyl alcohol, phenethyl alcohol, dehydroacetic acid, sorbic acid and the like.
Preferable examples of the antioxidant include sulfites, ascorbic acid and the like.
Preferable examples of the colorant include tar pigments, caramel, iron sesquioxide, titanium oxide, riboflavins and the like.
Suitable examples of sweeteners include glucose, fructose, invert sugar, sorbitol, xylitol, glycerin, simple syrup and the like.
Hereinafter, the present invention will be described in more detail with reference to examples, but these do not limit the scope of the present invention.

Example 1: Binding inhibition test The binding inhibition ability of aprepitant between SFN and USP8 or SKP1 was examined.
(Materials and methods)
1. 1. cell
As the cells, a cultured cell line A549 established from well-differentiated lung adenocarcinoma was used. A549 was cultured in D-MEM / F12 medium supplemented with 10% fetal bovine serum on a collagen coated dish with a diameter of 10 cm in a CO ₂ incubator under 37 ° C. and 5% CO ₂ concentration conditions. ..
2. 2. Aprepitant treatment
Aprepitant was added to cells cultured in a culture dish having a final concentration of 10 μM and a diameter of 10 cm. After 1 hour, the medium is removed, the cells are rinsed with PBS, and then 500 μl of M-PERTM Mammalian Protein Extraction Reagent (PIERCE, Rockford, IL), 3 μl of Protease inhibitor cocktail (PIERECE), and 5 μl of Phosphatase inhibitor cocktail (PIERECE) are added. , Protein was extracted. The extracted protein was quantified using the BCA Protein Assay Reagent Kit (PIERECE).
3. 3. Immunoprecipitation
For immunoprecipitation, anti-human SFN mouse monoclonal antibody (GeneTex, Inc., San Antonio, TX) and protein A magnetic beads were added at 500-fold dilution of the extracted protein solution and reacted at 4 ° C. overnight. The beads were then precipitated in a magnetic rack to remove the supernatant, and the immunoprecipitated product eluted from the precipitated beads was analyzed by Western blotting.
4. Western blot
Western blots were denatured from the immunoprecipitated product at 95 ° C. for 5 minutes and then electrophoresed using 10% Mini-PROTEAN TGX Precast Gels (Bio-Rad Laboratories, Hercules, CA, USA). The protein was then transferred to the PVDF membrane by the iBlotTM gel transfer system (Life Technologies). The primary antibody is an anti-human SFN mouse monoclonal antibody (GeneTex, Inc., San Antonio, TX; 200-fold diluted) and the anti-human SKP1 mouse monoclonal antibody (BD Bioscience, San Jose, CA; 500-fold diluted), and the secondary antibody is HRP. A labeled anti-mouse IgG secondary antibody (PIERCE; 500-fold diluted) was used. The protein band was detected by the ChemiDoc Touch Imaging System (Bio-Rad Laboratories) using SuperSignal West Femto® extended duration substrate (PIERCE).

(result)
The results are shown in FIG. It can be seen that the addition of aprepitant reduces USP8 and SKP1 that bind to SFN, and that aprepitant inhibits the binding between SFN and USP8 and the binding between SFN and SKP1.
Similarly, the binding inhibitory ability of ticagrelor, ZM241385, ezetimibe and chlorhexidine was investigated. The results are shown in Table 1.

Example 2: Cell proliferation inhibitory test Aprepitant was examined for its proliferation inhibitory effect on cancer cells.
(Materials and methods)
1. 1. cell
As the cells, a cultured cell line A549 established from well-differentiated lung adenocarcinoma was used. A549 was cultured in D-MEM / F12 medium supplemented with 10% fetal bovine serum on a collagen coated dish with a diameter of 10 cm in a CO ₂ incubator under 37 ° C. and 5% CO ₂ concentration conditions. ..
2. 2. Inhibitor preparation
Each inhibitor used DMSO as a solvent and was miscible with a vortex mixer.
3. 3. Cell proliferation test by WST-8
The day before compound addition, 6,000 cells / 100 μl were added to each well of a 96-well cell culture plate and cultured overnight in a CO ₂ incubator. After that, each inhibitor was added at a final concentration of 0.1, 1 and 10 μM, and the cells were cultured again in a CO ₂ incubator for 48 hours. After each incubation time, 10 μl of WST-8 reagent was added, and after incubation in the presence of CO ₂ for 1 hour, the absorbance at 450 nm was measured. As a control, cells to which 1 μl of DMSO was added were used, and the absorbance of these cells when WST-8 was added was set to proliferation ability 1.
(result)
The results are shown in FIG.
Similarly, the cell proliferation inhibitory effects of ticagrelor, ZM241385, ezetimibe and chlorhexidine were investigated.
The concentration of each inhibitor was 10 μM in which the cell proliferation inhibitory effect of aprepitant was confirmed at any elapsed time.
Similar to aprepitant, ticagrelor, ZM241385, ezetimibe and chlorhexidine were also confirmed to have cell proliferation inhibitory effects.

According to the present invention, cancer can be effectively treated without the need for companion diagnostics. In the case of lung adenocarcinoma, SFN is upregulated from the stage of early invasive lung adenocarcinoma, so for early lung adenocarcinoma for which no treatment method other than surgical excision has been established so far. Effective treatment can be provided.

Claims (2)

(I) An anticancer agent containing , as an active ingredient, a compound that inhibits the binding of Stratifin to at least one of (ii) SKP1 and USP8, wherein the compound is a compound represented by the following formula or a drug thereof. An anti-cancer drug that is an acceptable salt and the cancer is early stage lung adenocarcinoma .

The anticancer agent according to claim 1, wherein the compound is aprepitant or a pharmaceutically acceptable salt thereof.

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