mTOR inhibitors

Abstract

The present invention relates to an mTOR inhibitor containing adefovir or tenofovir, or a prodrug thereof, or a pharmaceutically acceptable salt thereof.

Description

[Technical field]

[0001] The present invention relates to mTOR inhibitors. [Background technology]

[0002] Adefovir (ADV) and tenofovir (TDF) are known as drugs used to treat hepatitis B virus (HBV) infection. Non-Patent Document 1 discloses that serum IFN-λ3 levels are high only in hepatitis B patients taking ADV or TDF, and that ADV or TDF induces IFN-λ3 in intestinal cells using an in vitro system. It also discloses that the induced IFN-λ3 induces IFN-stimulated genes (ISG) in hepatocytes and exhibits an inhibitory effect on HBs antigen production.

[0003] On the other hand, in some patients with hepatitis B virus infection, the autoimmune system becomes activated in response to the infection, causing liver inflammation. As the inflammation becomes chronic, it progresses to chronic hepatitis B and cirrhosis, and ultimately to liver cancer. Non-patent literature 2 presents the results of a cohort study conducted in Korea regarding the risk of hepatocellular carcinoma in patients treated for chronic hepatitis B. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Gut. 2018 67(2):362-371 [Non-Patent Document 2] JAMA Oncol. 2019 5(1):30-36 Summary of the Invention [Problem to be solved by the invention]

[0005] Although treatment of hepatitis B virus infection with ADV or TDF may be able to prevent the onset of liver cancer, it has not been known that ADV or TDF has a direct antitumor effect.

[0006] An object of the present invention is to provide a novel compound having an antitumor effect. [Means for solving the problem]

[0007] The present inventors have conducted extensive research to solve the above problems and have found that ADV and TDF have antitumor effects due to their mTOR inhibitory effect and Akt phosphorylation inhibitory effect, thereby completing the present invention.

[0008] That is, the present invention is as follows. (1) An mTOR inhibitor comprising adefovir or tenofovir or a prodrug thereof, or a pharma- ceutically acceptable salt thereof. (2) An Akt phosphorylation inhibitor comprising adefovir or tenofovir, or a prodrug thereof, or a pharma- ceutically acceptable salt thereof. (3) An antitumor agent comprising adefovir or tenofovir, or a prodrug thereof, or a pharma- ceutically acceptable salt thereof. (4) The antitumor agent according to (3), wherein the tumor is not caused by hepatitis. (5) The antitumor agent according to (4), wherein the hepatitis is hepatitis B. (6) The antitumor agent according to any one of (3) to (5), which is to be administered to a patient not infected with a hepatitis virus. (7) The antitumor agent according to any one of (3) to (6), which is used in combination with another antitumor agent. (8) An mTOR inhibitor and / or an Akt phosphorylation inhibitor for treating tumors and / or cancer, preferably for administration to a patient, more preferably a human patient, comprising a therapeutically effective amount / therapeutically effective amount of adefovir or tenofovir or a prodrug thereof, or a pharma- ceutically acceptable salt thereof. The tumor and / or cancer is preferably a tumor and / or cancer not caused by hepatitis, and may be a tumor and / or cancer not caused by hepatitis B. The patient or human patient may be for administration to a patient or human patient not infected with a hepatitis virus. It may be used in combination with other antitumor agents. (9) An antitumor agent for treating tumors and / or cancer, preferably for administration to a patient, more preferably a human patient, comprising a therapeutically effective amount / a therapeutically effective amount of adefovir or tenofovir or a prodrug thereof, or a pharma- ceutically acceptable salt thereof, which treats tumors and / or cancer by inhibiting mTOR and / or inhibiting Akt phosphorylation. The tumor and / or cancer is preferably a tumor and / or cancer not caused by hepatitis, and may be a tumor and / or cancer not caused by hepatitis B. The patient or human patient may be for administration to a patient or human patient not infected with a hepatitis virus. It may be used in combination with other antitumor agents. (10) The mTOR inhibitor and / or Akt phosphorylation inhibitor containing adefovir or tenofovir or a prodrug thereof, or a pharmaceutically acceptable salt thereof, may be used in a method of treating tumors and / or cancer by administering in a therapeutically effective amount / therapeutically effective amount to a subject, preferably a patient, more preferably a human patient in need thereof. Also, the adefovir or tenofovir or a prodrug thereof, or a pharmaceutically acceptable salt thereof, may be used in a method of treating tumors and / or cancer by administering in a therapeutically effective amount / therapeutically effective amount to a subject, preferably a patient, more preferably a human patient in need thereof, may be used in a method of inhibiting mTOR and / or inhibiting Akt phosphorylation, may be used in a method of treating tumors and / or cancer by inhibiting mTOR and / or inhibiting Akt phosphorylation. The tumor and / or cancer is preferably a tumor and / or cancer not caused by hepatitis, and may be a tumor and / or cancer not caused by hepatitis B. The patient or human patient may be for administration to a patient or human patient not infected with a hepatitis virus. It may be used in combination with other antitumor agents. (11) adefovir or tenofovir or a prodrug thereof, or a pharma- ceutically acceptable salt thereof, for inhibiting mTOR and / or inhibiting Akt phosphorylation; adefovir or tenofovir or a prodrug thereof, or a pharma- ceutically acceptable salt thereof, for treating tumors and / or cancer; adefovir or tenofovir or a prodrug thereof, or a pharma- ceutically acceptable salt thereof, for treating tumors and / or cancer by inhibiting mTOR and / or inhibiting Akt phosphorylation; Use for the manufacture of pharmaceutical formulations and / or compositions containing adefovir or tenofovir or a prodrug thereof, or a pharma- ceutically acceptable salt thereof, for use in treating tumors and / or cancer, for use in inhibiting mTOR and / or inhibiting Akt phosphorylation, or for use in treating tumors and / or cancer by inhibiting mTOR and / or inhibiting Akt phosphorylation; and Use / utilization of adefovir or tenofovir or a prodrug thereof, or a pharma- ceutically acceptable salt thereof, for inhibiting mTOR and / or inhibiting Akt phosphorylation, for treating tumors and / or cancer, or for treating tumors and / or cancer by inhibiting mTOR and / or inhibiting Akt phosphorylation. Adefovir or tenofovir or their prodrugs, or their pharma- ceutically acceptable salts, may be administered in a therapeutically effective amount to a subject, preferably a patient, more preferably a human patient, in need thereof. The tumor and / or cancer is preferably a tumor and / or cancer not caused by hepatitis, and may be a tumor and / or cancer not caused by hepatitis B. The patient or human patient may be for administration to a patient or human patient not infected with a hepatitis virus. It may be used in combination with other antitumor agents. Effect of the Invention

[0009] According to the present invention, a new compound having an antitumor effect can be provided. [Brief description of the drawings]

[0010] [Figure 1]The results of a study on the modulation of cytokine profiles in peripheral blood lymphocytes (PBMCs) are shown. (A) The average results of three healthy adults (HVs) are shown. (B) The results of enzyme-linked immunosorbent assay (ELISA) of seven HVs are shown. (C) In the examples, ADV refers to adefovir pivoxil and TDF refers to tenofovir disoproxil fumarate. Dose-dependent production of interleukin (IL)-12p70, IL-10 and tumor necrosis factor (TNF)-α by ADV and TDF was observed (1:3 dilution). n is at least 3. (D) The results are shown using PBMCs from a patient with chronic hepatitis B (CHB; 44-year-old male, HBeAg+, genotype A). (E) The results are shown using PBMCs from a patient with liver cirrhosis (LC) B (68-year-old female, HBeAg-, genotype C). (F) Results of PBMCs from HV (n=7), asymptomatic carriers (n=8), CH patients (n=10), and LC patients (n=5) treated with 500 nM ADV (** p <0.01 and *** p <0.001). [Diagram 2] The results of a study on the induction of synergistic LPS-stimulated IL-12p70 production in combination with interferon (IFN)-α are shown. Interferon (IFN)-α alone was at 10, 100, and 1,000 U / mL. [Diagram 3] Time course of phosphorylation of mitogen-activated protein kinase (MAPK)-associated proteins after lipopolysaccharide (LPS) stimulation. CD14+ monocytes positively selected using magnetic activated cell sorting (MACS) were stimulated with 100ng / mL LPS, and cells were harvested at each time point. Western blot analysis was performed using pMSK (phosphorylated-pediatric musculoskeletal)-1, pERK (extracellular signal-regulated kinase), and pp38 as primary antibodies. Phosphorylation of these proteins peaked 30 minutes after LPS stimulation. [Figure 4]Results of a study of mTOR pathway inhibition are shown. (A) CD14+ monocytes were positively selected by magnetic activated cell sorting (MACS) and pretreated with nucleic acid analogs for 2 hours, followed by stimulation with 100 ng / mL lipopolysaccharide (LPS). IL-10 was measured in the supernatant by enzyme-linked immunosorbent assay (ELISA). The purity of CD14+ monocytes was >95%. (B) CD14+ monocytes were treated with nucleic acid analogs, 10 μM SB202190 or 10 μM U0126 for 2 hours, followed by stimulation with 100 ng / mL LPS, and cells were harvested 30 minutes after LPS stimulation for Western blot analysis. (C) CD14+ monocytes were treated with nucleic acid analogs, 100 nM rapamycin, or 5 μM wortmannin for 2 hours, followed by stimulation with 100 ng / mL LPS. Cells were harvested 2 hours after LPS stimulation and subjected to Western blot analysis. (D) CD14+ monocytes were treated with the nucleic acid analog and subsequently stimulated with 100 ng / mL LPS (1:3 dilution). (E) CD14+ monocytes were treated with the nucleic acid analog for 2 hours and subsequently stimulated with 100 ng / mL LPS. Cells were harvested 2 hours after LPS stimulation and subjected to Western blot analysis (phosphorylated glycogen synthase kinase [pGSK]-3β). [Diagram 5] Time course of mammalian target of rapamycin (mTOR)-associated protein phosphorylation following lipopolysaccharide (LPS) stimulation. CD14+ monocytes positively selected using magnetic activated cell sorting (MACS) were stimulated with 100ng / mL LPS and cells were harvested at each time point. Western blot analysis was performed using pAkt1, pmTOR and pp70S6K as primary antibodies. Phosphorylation of these proteins peaked 2 hours after LPS stimulation. [Figure 6] 1 shows the results of a study of cytokine modulation by mTOR inhibitors. [Figure 7]The results of a test for inhibition of Akt translocation to the cell membrane are shown. (A) CD14+ monocytes were pretreated with nucleic acid analogs or 5 μM wortmannin (Wort) for 2 hours and then stimulated with 100 ng / mL LPS. The cells were harvested. Two hours after LPS stimulation, Western blot analysis was performed for phosphorylation of upstream proteins of Akt (phosphatidylinositol-3 kinase [PI3K] and phosphoinositide-dependent kinase 1 [PDK1]). (B) HT-1080 cells stably expressing Akt2PH-EGFP were treated with nucleic acid analogs for 2 hours, then fixed and probed with anti-phospho-Akt (Ser The results are shown stained with 473 and DAPI. Akt translocation to the plasma membrane (green) and Akt phosphorylation (red) were inhibited by ADV or TDF. Each inset shows a magnified image of the plasma membrane (Akt translocation; top, Akt phosphorylation; middle, confluence; bottom). (C) The interaction of Akt with nucleic acid analogues was tested using a surface plasmon resonance (SPR) biosensor. Akt inhibitor 2 (Akt-1 / 2) and bovine serum albumin (BSA) were used as positive and negative controls, respectively. (D) PBMCs were pretreated with various concentrations of Perifosine for 2 hours and then stimulated with 100 ng / mL LPS. After 24 hours of LPS stimulation, the supernatants were measured by enzyme-linked immunosorbent assay (ELISA). (E) PBMCs were treated with various concentrations of Perifosine, and the cells were harvested 2 hours after LPS stimulation, and Western blot analysis was performed. [Figure 8] A schematic diagram of the regulatory mechanism of LPS-mediated IL-10 production by ADV and TDF is shown. The cellular metabolites of ADV and TDF bind to Akt protein and inhibit the translocation of Akt to the cell membrane, and subsequently inhibit its phosphorylation upon LPS administration, thereby inhibiting LPS-mediated IL-10 production. TF stands for transcription factor. [Figure 9]Phosphatase and tensin homolog (PTEN) did not contribute to the inhibition of LPS-mediated IL-10 by ADV and TDF. CD14+ monocytes positively selected by magnetic activated cell sorting (MACS) were pretreated with nucleic acid analogs for 2 hours, and the cells were harvested without LPS stimulation. The cells were further fractionated into cytosolic and nuclear fractions, and the results of Western blot analysis of each fraction are shown. Poly ADP-ribose polymerase (PARP) is an internal control for nuclear proteins, and β-actin is an internal control for the cytosolic fraction. n is at least 3. [Figure 10] Mammalian target of rapamycin complex 2 (mTORC2) did not contribute to the inhibition of IL-10 by ADV and TDF. CD14+ monocytes positively selected by magnetic activated cell sorting (MACS) were transfected with control siRNA and rictor using a transfection kit. The expression levels of rictor, raptor, and β-actin were confirmed 48 hours after transfection. The cells were then pretreated with nucleic acid analogs for 2 hours, and then stimulated with 100 ng / mL LPS. IL-10 was measured in the supernatants by enzyme-linked immunosorbent assay (ELISA) 24 hours after LPS stimulation. [Figure 11]5′-AMP-activated protein kinase (AMPK) did not contribute to the inhibition of IL-10 by ADV and TDF. (A) PBMCs were pretreated with various concentrations of 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) for 2 h and then stimulated with 100 ng / mL LPS. 24 h after LPS stimulation, IL-12p70 and IL-10 in the supernatants were analyzed by enzyme-linked immunosorbent assay (ELISA). (B) CD14+ monocytes positively selected using magnetic activated cell sorting (MACS) were pretreated with various concentrations of AICAR for 2 h and stimulated with 100 ng / mL LPS. Cells were harvested 2 h after LPS stimulation and subjected to Western blot analysis. (C) CD14+ monocytes positively selected using MACS were transfected with control siRNA and AMPK using the Nucleofector kit. Forty-eight hours after transfection, cells were harvested and subjected to Western blot analysis. (D) CD14+ monocytes transfected with control siRNA or AMPK were pretreated with nucleic acid analogs for 2 hours and then stimulated with LPS for 24 hours. IL-10 was measured in the supernatants by enzyme-linked immunosorbent assay (ELISA). [Figure 12] The antitumor effects of entecavir (ETV) or TDF on various liver cancer cell lines are shown. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The mTOR (mammalian target of rapamycin) inhibitor of the present invention contains adefovir or tenofovir, or a prodrug thereof, or a pharma- ceutically acceptable salt thereof. Adefovir is a compound represented by the following formula (I), and hereinafter, adefovir or adefovir prodrug, or a pharma- ceutically acceptable salt thereof may be collectively referred to as "adefovir, etc." in this specification. Formula (I):

[0012] [ka]

[0013] Tenofovir is a compound represented by the following formula (II), and hereinafter, tenofovir or a prodrug of tenofovir, or a pharma- ceutically acceptable salt thereof may be collectively referred to as "tenofovir, etc." in this specification. Formula (II):

[0014] [ka]

[0015] The prodrugs of adefovir and tenofovir may be known prodrugs as prodrugs of a phosphate group, and are preferably known prodrugs as nucleic acid analog prodrugs. The prodrug is not particularly limited, but examples thereof include prodrugs in which the phosphate group is substituted with a pivoxil group, a disoproxil group, an alafenamide group, or the like.

[0016] Salts of adefovir and tenofovir or salts of prodrugs of adefovir and tenofovir include alkali metal salts, alkaline earth metal salts, ammonium salts, amino acid salts, organic acid salts and inorganic acid salts.

[0017] The mTOR inhibitor of the present invention preferably contains adefovir etc. and / or tenofovir etc. as an active ingredient. The adefovir etc. and / or tenofovir etc. is not particularly limited, but for example, adefovir pivoxil (also called adefovir dipivoxil), tenofovir alafenamide fumarate, and tenofovir disoproxil fumarate can be suitably used.

[0018] In the present invention, the term "as an active ingredient" means that it is contained as a main active ingredient, and so long as it contains adefovir etc. and / or tenofovir etc. as a medicinal ingredient (an ingredient that exerts a pharmacological effect), the content thereof is not particularly limited.

[0019] In the present invention, adefovir etc. and tenofovir etc. may be in the form of a hydrate or a solvate. In addition, adefovir, etc. and tenofovir, etc. may be isotope-labeled. The isotope is not particularly limited, but examples thereof include stable isotopes of phosphorus, carbon and hydrogen atoms such as 33P, 32P, 13C and 2H(D).

[0020] Since mTOR signaling is commonly activated in tumor cells, the mTOR inhibitor of the present invention exerts an antitumor effect by inhibiting mTOR.

[0021] The present invention provides an Akt phosphorylation inhibitor, which contains adefovir or tenofovir, a prodrug thereof, or a pharma- ceutically acceptable salt thereof. Akt is a kinase that is activated in a PI3K-dependent manner, and induces mTOR (mammalian target of rapamycin) by controlling downstream signals such as Bad and GSK-3. Therefore, the Akt phosphorylation inhibitor of the present invention suppresses the induction of mTOR, and thus exerts an antitumor effect.

[0022] In the present invention, mTOR inhibitors or Akt phosphorylation inhibitors can suppress the proliferation of cancer cells or sarcoma cells and induce cell death (apoptosis) by inhibiting the PI3K / Akt / mTOR pathway, and can therefore be used as antitumor agents.

[0023] The antitumor agent of the present invention contains adefovir or tenofovir, or a prodrug thereof, or a pharma- ceutically acceptable salt thereof. The tumor for which the antitumor agent of the present invention is used is not particularly limited, but examples thereof include hematopoietic tumors and solid cancers. Examples of hematopoietic tumors include acute leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia, polycythemia vera, malignant lymphoma, and myeloma. Examples of solid cancers include brain tumors, head and neck cancer, esophageal cancer, thyroid cancer, small cell lung cancer, non-small cell lung cancer, breast cancer, gastric cancer, gallbladder and bile duct cancer, liver cancer, pancreatic cancer, colon cancer, rectal cancer, ovarian cancer, chorioepithelial cancer, uterine cancer, cervical cancer, urothelial cancer, renal cell carcinoma, prostate cancer, testicular tumors, Wilms' tumor, malignant melanoma, neuroblastoma, osteosarcoma, Ewing's sarcoma, and soft tissue sarcoma.

[0024] The antitumor agent of the present invention is used for the purpose of preventing and / or treating cancer, and is preferably used for cancer treatment. In particular, it has the effect of shrinking or eliminating tumors, or suppressing tumor growth to a certain level or below.

[0025] The antitumor agent of the present invention preferably exerts a direct antitumor effect by inhibiting mTOR or inhibiting Akt phosphorylation, unlike drugs that can be used to prevent cancer by treating hepatitis B virus (HBV) infection, and can be used as an antitumor agent for tumors not caused by hepatitis. The hepatitis is not particularly limited, but is preferably hepatitis B, for which adefovir and / or tenofovir are used. The antitumor agent of the present invention can be used as an antitumor agent for tumors not caused by hepatitis, preferably not caused by hepatitis B. Tumors not caused by hepatitis, preferably not caused by hepatitis B, may be the above-mentioned hematopoietic tumors or solid cancers, but the agent may also be used as an antitumor agent for liver cancer not caused by hepatitis, preferably not caused by hepatitis B.

[0026] The mTOR inhibitor, Akt phosphorylation inhibitor, and antitumor agent (hereinafter sometimes referred to as "antitumor agent, etc.") of the present invention are used as drugs, pharmaceutical preparations, and / or pharmaceutical compositions, and can be administered orally or parenterally. For oral administration, known dosage forms for administration can be used, such as tablets, capsules, coated tablets, troches, liquids such as solutions or suspensions. Parenteral administration includes intravenous, intramuscular, or subcutaneous administration by injection; transmucosal administration into the nasal or oral cavity using powders, drops, sprays, aerosols, or the like; rectal administration using creams or suppositories, and transdermal administration using patches, liniments, gels, and the like. The route of administration is preferably oral or intravenous by injection.

[0027] In the present invention, the antitumor agent etc. may be used in combination with other antitumor agents. Other antitumor agents can be appropriately selected depending on the target tumor. The antitumor agent, etc. of the present invention preferably further regulates lipopolysaccharide (LPS)-mediated cytokine profile in peripheral blood lymphocytes (PBMCs). Specifically, the antitumor agent, etc. of the present invention inhibits LPS-induced interleukin (IL)-10 production and mutually induces LPS-mediated IL-12p70 and tumor necrosis factor (TNF)-α expression. In addition, the combination of IFN-α and the tumor agent, etc. synergistically enhances LPS-mediated IL-12p70 production in PBMCs. Here, downregulation of IL-10, which suppresses excessive immune responses, is expected to restore the functions of T cells and NK cells. Therefore, the antitumor agent etc. of the present invention not only has mTOR inhibitory activity and / or Akt phosphorylation inhibitory activity, but also functions as an IL-10 production inhibitor, which leads to enhancing the antitumor activity of other antitumor agents, and is therefore preferably used in combination with other antitumor agents.

[0028] The antitumor agent etc. of the present invention may be an mTOR inhibitor or an Akt phosphorylation inhibitor, or may be an IL-10 production inhibitor. The antitumor agent etc. of the present invention may be an mTOR inhibitor and an IL-10 production inhibitor, or may be an Akt phosphorylation inhibitor and an IL-10 production inhibitor, or may be an mTOR inhibitor, an Akt phosphorylation inhibitor, and an IL-10 production inhibitor. Furthermore, the antitumor agent etc. of the present invention may be an IL-12p70 production enhancer, a TNF-α production enhancer, an IL-12p70 expression inducer, or a TNF-α expression inducer. The antitumor agent etc. of the present invention may be an mTOR inhibitor and an IL-12p70 production enhancer and / or a TNF-α production enhancer, an Akt phosphorylation inhibitor and an IL-12p70 production enhancer and / or a TNF-α production enhancer, or an mTOR inhibitor, an Akt phosphorylation inhibitor, and an IL-12p70 production enhancer and / or a TNF-α production enhancer. The antitumor agent etc. of the present invention may be an mTOR inhibitor and an IL-12p70 expression inducer and / or a TNF-α expression inducer, an Akt phosphorylation inhibitor and an IL-12p70 expression inducer and / or a TNF-α expression inducer, or an mTOR inhibitor, an Akt phosphorylation inhibitor, and an IL-12p70 expression inducer and / or a TNF-α expression inducer. The antitumor agent etc. of the present invention may further be an IL-10 production inhibitor.

[0029] When an antitumor agent, etc. is used as a pharmaceutical preparation and / or pharmaceutical composition, in addition to the antitumor agent, etc. and other tumor agents, a suitable pharma- ceutical acceptable carrier well known to those skilled in the art may be included, as appropriate, depending on the administration form, etc. Pharmaceutically acceptable carriers include, but are not limited to, antioxidants, stabilizers, preservatives, flavoring agents, colorants, dissolving agents, solubilizing agents, surfactants, emulsifiers, antifoaming agents, viscosity modifiers, gelling agents, absorption enhancers, dispersants, excipients, and pH adjusters.

[0030] In the present invention, the dosage and administration schedule of adefovir etc. and / or tenofovir etc. as the antitumor agent etc. may be adjusted depending on the required amount, treatment method, degree of disease or need, etc. of each individual subject to be treated. The dosage can be determined specifically depending on the age, body weight, general health condition, sex, diet, administration time, administration method, excretion rate, drug combination, and medical condition of the patient, and may also be determined taking other factors into consideration. As the antitumor agent etc. of the present invention, adefovir etc. and / or tenofovir etc. are preferably contained in an amount effective for exerting an antitumor effect. The daily dosage of adefovir etc. and / or tenofovir etc. varies depending on the condition and body weight of the patient, the type of compound, the administration route, etc., but in the case of parenteral administration, it is usually about 0.01 to 1000 mg / person / day, preferably 0.1 to 500 mg / person / day, and in the case of oral administration, it is usually about 0.01 to 500 mg / person / day, preferably 0.1 to 100 mg / person / day.

[0031] The present invention may be a method for preventing or treating cancer, which comprises administering an antitumor agent or the like to a patient in need thereof. The patient in need thereof is not particularly limited, and is a mammal, preferably a human. The antitumor agent and the like of the present invention are preferably administered to patients who are not infected with hepatitis viruses. As the antitumor agent, adefovir, etc. and / or tenofovir, etc., is preferably administered in a therapeutically effective amount. In the present invention, adefovir etc. and / or tenofovir etc. have an antitumor effect, and therefore may be used for the prevention of cancer by administering them before the onset of cancer, or may be used for the treatment of cancer. EXAMPLES

[0032] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to the following examples. In addition, various modifications can be made without departing from the technical spirit of the present invention.

[0033] Lamivudine was purchased from Sigma-Aldrich (St Louis, Missouri, USA), and other nucleic acid analogs were purchased from Adooq Bioscience (Irvine, Calif., USA). Primary antibodies against phosphorylated ERK, p38 (Thr180 / Thy182), MSK-1 (Thr581), Akt (Ser 473), p70S6K (Thr389), 4E-BP1 (Thr37 / Thr46), c-fos (Ser32), GSK-3β (Ser9), PDK1 (Ser241), PTEN (Ser380 / Thr382 / 383), Akt, PARP, PTEN, and b-actin were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibodies against phosphorylated PIK3R1 / PIK3R3 were purchased from Thermo scientific (Rockford, IL, USA). Monoclonal antibodies and recombinant proteins of IL-10, IL-12, and TNF-α were purchased from R&D systems (Minneapolis, MN, USA).

[0034] <Separation of various lymphocyte cells> Peripheral blood lymphocytes (PBMCs) were isolated by the density gradient method from blood collected from healthy adults (HV) and patients with chronic hepatitis B. CD3+ T cells, CD19+ B cells, CD56+ NK cells, and CD14+ monocytes were isolated using magnetic antibodies, respectively.

[0035] <Cell culture> PBMCs or CD14+ monocytes were incubated with various concentrations of nucleic acid analogs (NUCs), rapamycin, wortmannin, or perifosine in RPMI containing 10% bovine serum for 2 h, and then stimulated with 100 ng / mL lipopolysaccharide (LPS). Supernatants or cells were harvested at the indicated times. The concentrations of NAs used were 50 mM, 500 nM, 500 nM, and 5 mM for lamivudine (LAM), adefovir (ADV), entecavir (ETV), and tenofovir (TDF), respectively, unless otherwise indicated. These concentrations were 10 times higher than the peripheral blood concentrations of each NA. This is because the portal vein concentration of orally administered drugs has been reported to be 10 times higher than the peripheral blood concentration.

[0036] <Cytokine measurement> Multiple cytokines in the culture supernatants were measured using the Bio-Plex human cytokine panel, and IL-10, IL-12, and TNF-α were also analyzed by ELISA.

[0037] <Separation of cell fractions> For the separation of cytoplasm and cell membrane, the MEM-PERTM Plus Cell Membrane Protein Extraction Kit was used and the protocol indicated by the product was followed. Briefly, after washing the collected cells, they were reacted with a buffer (which makes holes in the cell membrane) at 4°C for 10 minutes. After centrifugation at 16,000 g for 15 minutes, the supernatant was collected into a new tube (cytoplasmic component). The precipitate in the tube at the previous step (cell membrane) was washed once with the above buffer and then reacted with a cell membrane lysis buffer at 4°C for 30 minutes with occasional shaking. After that, after centrifugation at 16,000 g for 15 minutes, the supernatant was collected into a new tube (cell membrane component). The cytoplasmic and nuclear components were separated using a cytoplasmic / nuclear extraction kit. That is, after washing the collected cells, they were reacted with a buffer (which makes holes in the cell membrane) at 4°C for 10 minutes, then centrifuged at 16,000 g for 15 minutes, and the supernatant was collected into a new tube (cytoplasmic component). The precipitate in the tube at the previous step (cell membrane + nucleus) was washed once with the above buffer and then reacted with a nuclear lysis buffer at 4°C for 30 minutes with occasional shaking. After that, after centrifugation at 16,000 g for 15 minutes, the supernatant was collected into a new tube (nuclear component).

[0038] <Gene Silencing by siRNA> Chemically synthesized nucleotide siRNA containing control siRNA was obtained from Santa Cruz (Dallas, TX, USA). The cells were transfected with siRNA using a Nucleofector kit (Lonza, Cologne, Germany).

[0039] <Western Blotting> The cells were incubated with blue-loading buffer for 30 minutes at 4℃, centrifuged at 16,000g for 15 minutes, and the supernatant was collected. The same amount of protein was loaded onto an SDS-PAGE gel and then transferred to a membrane. The membrane with the transferred protein was reacted with blocking buffer, and the primary antibody was diluted in the same solution and reacted overnight at 4℃. After washing the membrane, it was reacted with secondary antibody for 1 hour, and after further washing, it was reacted with Western Lighting ECL Pro NEL 12001EA, and the bands were detected with ImageQuant LAS 4000.

[0040] <Fluorescent immunochemical staining> HA-tagged AKT2-PH-EGFP was inserted into pCSII-EF1α-MCS-IRES2-Blasticidin lentivirus vector. HT1080 cells were infected with the lentivirus and cultured in DMEM containing 5mg / ml blastidine. To observe intracellular trafficking of PH-EGFP, phosphorylated Akt, and Akt-PH-EGFP, HT1080 cells expressing each were cultured on cover glasses with DMSO, LAM, ADV, ETV, or TDF for 2 hours, and then fixed with 3.7% formalin. Then, phosphorylated AKt (S473) and DAPI (nuclear staining) were stained and observed under a microscope.

[0041] <Surface plasmon resonance analysis> The binding affinity of the peptides to the compounds was analyzed using a surface plasmon resonance biosensor (Biacore X100, GE Healthcare). Akt1 or BSA was immobilized on the surface of a CM5 sensor chip using an amine coupling kit (GE Healthcare). Different concentrations of compounds diluted in phosphate buffered saline (10 mM phosphate buffer, 2.7 mM KCl, 137 mM NaCl, 0.05% surfactant P20, pH 7.4) containing 5% DMSO were injected over the sensor chip at a flow rate of 20 μL / min for 120 s at 25 °C, followed by application of compound-free buffer for 180 s. The bulk effect of DMSO was subtracted using a reference flow cell, and kinetic parameters were determined by analyzing the data using Biacore X100 evaluation software (GE Healthcare).

[0042] <Statistical analysis> Pearson's chi-square test and Fisher's exact test were used to compare nonparametric categorical data. One-way analysis of variance was used for analysis of variance. All tests were performed using the IBM Statistical Package for Social Sciences, Statistics Desktop for Japan, V.19.0 (IBM JAPAN), and a p-value <0.05 was considered statistically significant.

[0043] Modulation of cytokine profiles in LPS-stimulated PBMCs To test whether the NAs affect LPS-induced cytokine production, PBMCs from three HVs were pretreated ex vivo with the NAs for 2 h and subsequently stimulated with LPS and analyzed for cytokine production using the BioPlex cytokine assay. Of the 15 cytokines tested, granulocyte-colony stimulating factor (G-CSF), IL-12, IL-10, interferon (IFN)-γ, granulocyte-macrophage (GM)-CSF, IL-13, IL-17, IL-1β, IL-2, IL-4, IL-6, IL-7, IL-8, monocyte chemotactic and activating factor (MCAF), and tumor necrosis factor (TNF)-α., the NAs altered LPS-induced production of three cytokines (IL-10, IL-12p70, and TNF-α). ADV or TDF significantly induced IL-12p70 and TNF-α and inhibited IL-10, whereas LAM and ETV only showed effects similar to those of DMSO (FIG. 1A). PBMCs derived from other HVs were treated similarly, and IL-12p70, IL-10, and TNF-α were measured using ELISA, showing results similar to those of the BioPlex assay (Figure 1B). These cytokine modulations were ADV or TDF dose-dependent (Figure 1C). Similar patterns were observed using PBMCs from HBV patients with chronic hepatitis (CH; Figure 1D) or liver cirrhosis (LC; Figure 1E). When cytokine production in PBMCs obtained from different stages of HBV infection was compared, no significant differences were observed among HV, asymptomatic carriers, and CH patients. The production of all cytokines in LC patients was lower than that in HV, asymptomatic carriers, or CH patients (Figure 1F).

[0044] Synergistic induction of LPS-stimulated IL-12p70 production by combination with IFN-α PBMCs were treated ex vivo with nucleic acid analogs, IFN-α, and LPS, and IL-10 and IL-12p70 supernatant levels were measured. When combined with ADV or TDF, IFN-α synergistically enhanced LPS-mediated IL-12p70 production (Figure 2).

[0045] Inhibition of the mTOR pathway CD14+ monocytes were stimulated with LPS and analyzed for phosphorylation of MAPK-related proteins (pp38, pERK, and pMSK-1), which were maximally phosphorylated 30 min after LPS stimulation (Figure 3). Under these conditions, U0126 and SB202198 inhibited the phosphorylation of ERK and MSK-1, respectively, whereas the nucleic acid analogs did not inhibit the phosphorylation of these proteins (FIG. 4B). mTOR-associated proteins were maximally phosphorylated 2 h after LPS stimulation (Fig. 5 ). Under these conditions, ADV and TDF inhibited the phosphorylation of Akt and p70S6K (FIG. 4C). ADV and TDF dose-dependently inhibited the phosphorylation of Akt (Fig. 4D). ADV and TDF also inhibited the phosphorylation of GSK-3β (Fig. 4E).

[0046] Cytokine regulation by mTOR inhibitors The mTOR inhibitors rapamycin (Fig. 6A) and wortmannin (Fig. 6B) dose-dependently inhibited LPS-mediated IL-10 production and induced LPS-mediated IL-12p70 production, indicating that LPS-mediated cytokine regulation by ADV and TDF was due to inhibition of the mTOR pathway.

[0047] Inhibition of Akt translocation to the plasma membrane We analyzed the phosphorylation of several proteins upstream of Akt, such as phosphatidylinositol-3 kinase and phosphoinositide-dependent kinase 1. ADV and TDF did not inhibit the phosphorylation of these proteins (FIG. 7A). Stable Akt2-PH-EGFP-expressing HT1080 cells were treated with the nucleic acid analogs for 2 h and examined by confocal microscopy, and it was found that ADV and TDF significantly inhibited the translocation of Akt-PH to the plasma membrane, which was consistent with the decreased phosphorylation level of Akt in HT1080 cells (Fig. 7B). The binding affinity of Akt for ETV, TDF, or tenofovir monophosphate (TFV) was analyzed using a surface plasmon resonance biosensor, and TFV bound to Akt protein in a dose-dependent manner (Figure 7C). When PBMCs were pretreated for 2 h with perifosine, which is known to inhibit the translocation of Akt to the cell membrane, and then stimulated with LPS, the production levels of IL-10 and IL-12p70 were dose-dependently decreased and increased, respectively (Fig. 7D).Perifosine also dose-dependently inhibited the phosphorylation of Akt (Fig. 7E).

[0048] No inhibition of upstream molecules of mTOR Phosphatase tensin homolog (PTEN), mTOR complex 2, and 5'-AMP-activated protein kinase (AMPK) are known to inhibit the mTOR pathway. ADV and TDF did not affect PTEN or pPTEN expression levels in LPS-treated CD14+ monocytes (Figure 9). The decrease in ADV- and TDF-induced IL-10 levels was not restored by silencing Rictor (FIG. 10). The AMPK inducer 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) inhibited IL-10 production and induced IL-12p70 when incubated with LPS-stimulated PBMCs (FIG. 11A). However, AICAR enhanced Akt phosphorylation in a dose-dependent manner (Fig. 11B).IL-10 production in PBMCs with AMPK silencing was not restored (Fig. 11C, D). These results indicated that PTEN, mTOR complex 2, and AMPK were not involved in the inhibition of IL-10 production by ADV and TDF.

[0049] Antitumor effects of ADV and TDF in hepatocellular carcinoma cell lines and animal models We investigated whether ETV or TDF exhibited antitumor effects by measuring the intracellular ATP levels using CellTiter-Glo (Promega) after culturing various hepatocellular carcinoma cell lines (HepG2, PLC / PRF / 5, Huh7, Hep3B, SKHep-1, HLE) with ETV or TDF for 72 hours. In all cell lines, TDF significantly reduced cell viability in a dose-dependent manner, whereas ETV had no effect (Figure 12).

[0050] After culturing human hepatoma cell line (HepG2) with LAM, ETV, ADV, or TDF, phosphorylation of Akt in the mTOR pathway and its downstream proteins S6K and 4-EBP were analyzed by Western blot. ADV and TDF dose-dependently suppressed phosphorylation of the above proteins, but LAM and ETV did not change it.

[0051] Using mouse hepatoma cell lines (MHCF1, MHCF5) and colon cancer cell line (CMT-93), the mTOR inhibitory effects of ETV and TDF were analyzed by Western blotting to measure the phosphorylation of Akt and its downstream GSK-3β (S9).The phosphorylation of mTOR-related proteins was suppressed in the human hepatoma cell line HepG2 cells, but no effect of TDF was observed in mouse hepatoma cell lines and colon cancer cell lines (data not disclosed).

[0052] DMSO (negative control), ETV, ADV, and TDF were intraperitoneally administered at 90 mg / kg each to 6-week-old immunodeficient mice (NOD-SCID) (5 mice per group). One week after the start of administration, HepG2 (5 × 10 6) were administered subcutaneously, and the subcutaneous tumor diameter was measured 6 weeks later. Each drug was administered intraperitoneally every 3 days from the start to the end of observation. There was no difference in tumor diameter between the ETV group and the DMSO group, but tumor diameter was suppressed with statistically significant differences in the ADV and TDF groups (p<0.05 or p<0.005 vs. the ETV group or DMSO group, data not disclosed). In one case of the TDF group, the tumor did not grow at all, demonstrating an extremely strong antitumor effect. These results suggest that ADV and TDF have antitumor effects in hepatocellular carcinoma cell lines both in vitro and in vivo, which are based on their inhibitory effect on the mTOR pathway observed in monocytes. Furthermore, their antitumor effects on carcinogenesis are thought to be based on their inhibitory effect on Akt phosphorylation.

Claims

1. An mTOR inhibitor for treating liver cancer comprising adefovir or tenofovir or a prodrug thereof, or a pharma- ceutically acceptable salt thereof, wherein the prodrug is adefovir or tenofovir, and the phosphate group of the adefovir or tenofovir is replaced with a pivoxil group, a disoproxil group, or an alafenamide group.

2. An Akt phosphorylation inhibitor for treating liver cancer, comprising adefovir or tenofovir or a prodrug thereof, or a pharma- ceutically acceptable salt thereof, wherein the prodrug is adefovir or tenofovir, and the phosphate group of the adefovir or tenofovir is replaced with a pivoxil group, a disoproxil group, or an alafenamide group.

3. An antitumor agent comprising adefovir or tenofovir or a prodrug thereof, or a pharma- ceutically acceptable salt thereof, wherein the phosphate group of adefovir or tenofovir is replaced with a pivoxil group, a disoproxil group, or an alafenamide group, wherein the tumor is liver cancer.

4. The antitumor agent according to claim 3, wherein the tumor is not caused by hepatitis.

5. The antitumor agent according to claim 4, wherein the hepatitis is hepatitis B.

6. The antitumor agent according to any one of claims 3 to 5, which is for administration to a patient not infected with a hepatitis virus.

7. The antitumor agent according to any one of claims 3 to 6, for use in combination with another antitumor agent.

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