KR20210107548A - Pharmaceutical Composition for Preventing or Treating Cancer Comprising mTOR pathway inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active component. A composition of the present invention inhibits the growth of cancer cells and exhibits the efficacy of killing cancer cells, thereby being able to be used as an anticancer agent.

Description

Pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active ingredient {Pharmaceutical Composition for Preventing or Treating Cancer Comprising mTOR pathway inhibitor}

The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active ingredient.

Cancer is the most common and serious disease that threatens human health, and research and development of anticancer drugs is important for prolonging the lifespan of cancer patients. In recent years, cancer treatment methods have made a lot of progress due to the rapid development of oncogenomics and molecular pharmacology and the development of new anticancer drugs, but there is still a need for the development of new therapeutic agents. Colorectal cancer is a malignant tumor that occurs in the appendix, colon, and rectum, and occurs in the mucous membrane, the innermost surface of the large intestine. Colorectal cancer is the second most common cancer in Korea after gastric cancer, and its incidence has increased sharply with the westernization of eating habits. Its rate of increase continues to increase.

Treatment of colorectal cancer can be divided into surgical resection, chemotherapy, and radiation therapy. Polyps, which are the pre-stage of colorectal cancer, or early colon/rectal cancer limited to polyps, can be treated with polypectomy. In the treatment of colorectal cancer, since Fluorouracil (5-FU) developed in the 1970s, five anticancer drugs such as irinotecan, oxaliplatin, capecitabine, TAS-102, and epithelial cell growth cetuximab, Panitu, a targeted anticancer agent that targets epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), or vascular endothelial growth factor receptor (VEGF-R) Five targeted anticancer drugs, including panitumumab, bevacizumab, aflibercept, and regorafenib, have been approved by the US FDA and are being used. However, there is still an urgent need to develop a treatment for colorectal cancer with excellent anticancer effect, stability, and absorption into the body.

mTOR (mechanistic target of rapamycin) protein is a serine/threonine kinase and is a key signaling factor that regulates cell growth, cell cycle, protein synthesis and glucose metabolism. In particular, as a key protein for regulating cell growth signaling, its activity is abnormally increased in 30% of solid cancer cells, and it is known that these emtors and higher-level factors (PI3K, Akt) are the most changed in cancer cells. Activation of emtor signals in cancer is caused by mutations in the emtor gene itself, high-level oncogenes (PI3K, Akt) that enhance the activity of emtor, and mutations in tumor suppressor factors (eg PTEN, TSC1/2). . Therefore, inhibition of emtor-associated signaling may induce cell autophagy along with protein synthesis inhibition, lipid synthesis inhibition, and cell growth inhibition, leading to cell death. Therefore, there is a need for the development of an emtor-related signaling inhibitor for the inhibition of cancer cell growth and cancer cell death.

Lomitapide for the treatment of hypercholesterolemia. Expert Opin Pharmacother. 2017 Aug;18(12):1261-1268. Efficacy and Safety of Lomitapide in Hypercholesterolemia. Am J Cardiovasc Drugs. 2017 Aug;17(4):299-309.

The present inventors made intensive research efforts to develop compounds for inhibiting cancer cell growth and killing cancer cells. As a result, the present invention was completed by identifying that the composition comprising an mTOR signaling inhibitor as an active ingredient is effective in treating cancer.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active ingredient.

According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active ingredient.

The emtor signal transduction inhibitor refers to a substance that inhibits the activity of emtor itself or inhibits the activity of an upper-level factor that increases the activity of emtor. The target whose activity is inhibited by the emtor signaling inhibitor may include, but is not limited to, emtor, PI3K, Akt, S6K, and S6.

In one embodiment of the present invention, the emtor signaling inhibitor is lomitapide.

Lomitapide (trade names Jaxtapid (US), Lojuxta (EU)) is a rare disease treatment developed by Aegerion Pharmaceuticals and is used as a cholesterol lowering agent in the treatment of familial hypercholesterolemia. On December 21, 2012, the U.S. Food and Drug Administration (FDA) reported that LDL cholesterol, total cholesterol, apolipoprotein B, and non-high-density lipoprotein Lomitapide was approved as a drug to reduce high-density HDL) cholesterol.

The IUPAC name of lomitapide is N- (2,2,2-trifluoroethyl)-9-[4-[4-[[[4'-(trifluoromethyl)[1,1'-biphenyl] ] 2-yl] carbonyl] amino] -1-piperidinyl] butyl] -9 H - fluoren-9-carboxylic a copy polyimide

In one embodiment of the present invention, the lomitapide is represented by the following formula (I).

[Formula I]

In one embodiment of the present invention, the emtor signaling inhibitor is lomitapide, a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

In one embodiment of the present invention, the cancer is a solid cancer.

As used herein, the term "solid cancer" has characteristics distinguishing it from blood cancer, and includes bladder, breast, intestine, kidney, lung, liver, brain, esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, prostate and skin. It is a cancer consisting of a mass caused by abnormal cell growth in various solid organs such as

In one embodiment of the present invention, the solid cancer is melanoma, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral lymphoma, oligodendroglioma, ependymoma, brain stem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal cavity / Sinus cancer, nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, Biliary tract cancer, pancreatic cancer, small intestine cancer, colorectal cancer, rectal cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, It is selected from the group consisting of female external genital cancer, female urethral cancer, bone tumor, duodenal cancer, fibrosarcoma, and skin cancer, but is not limited thereto.

In one embodiment of the present invention, the cancer is a hematologic cancer.

As used herein, the term “hematologic cancer” refers to cancer occurring in components constituting blood, and refers to malignant tumors occurring in blood, hematopoietic organs, lymph nodes, lymphatic organs, and the like.

In one embodiment of the present invention, the hematologic cancer is acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute monocytic leukemia, multiple myeloma, Hodgkin's lymphoma, and non-Hodgkin's lymphoma. is selected from, but not limited thereto.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the active ingredient, lomitapide, a pharmaceutically acceptable salt thereof, or an optical isomer thereof.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are commonly used in formulation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. it's not going to be

The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, and the like, in addition to the above components. Suitable pharmaceutically acceptable carriers and agents are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, for example, intrathecal administration, intravenous administration, subcutaneous administration, intradermal administration, intramuscular administration, intraperitoneal administration, intrasternal administration, intratumoral administration, intranasal administration , intracerebral administration, intracranial administration, intrapulmonary administration, rectal administration, etc., but is not limited thereto.

A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration mode, age, weight, sex, pathological condition, food, administration time, administration route, excretion rate and response sensitivity of the patient, An ordinarily skilled physician can readily determine and prescribe an effective dosage (a pharmaceutically effective amount) for the desired treatment or prophylaxis. According to a preferred embodiment of the present invention, the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg.

As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to prevent or treat the above-mentioned diseases.

As used herein, the term “prevention” refers to the prevention or protective treatment of a disease or disease state. As used herein, the term “treatment” refers to reduction, suppression, sedation or eradication of a disease state.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by a person of ordinary skill in the art to which the present invention pertains. or may be prepared by incorporation into a multi-dose container. At this time, the dosage form may be prepared in various ways such as oral medicine, injection, etc., in the form of a solution, suspension or emulsion in oil or aqueous medium, or in the form of an extract, powder, suppository, powder, granule, tablet or capsule, dispersant or stable Additional topics may be included.

According to an embodiment of the present invention, the pharmaceutical composition comprising lomitapide of the present invention has the effect of inhibiting the emtor signaling process and increasing the expression of proteins involved in the autophagy mechanism of cells.

As used herein, the term “autophagy” is a destructive process that naturally decomposes unnecessary or non-functional cellular components in the control process of cells.

The autophagy-related genes include ULK1, mTOR, ATG family, DDIT4, and the like.

ULK1 is a gene encoding a protein kinase required for the initiation of autophagy, and ULK1 activity is suppressed under conditions of high mTOR protein activity. When mTOR activity is inhibited, the increase in ULK1 activity directs it to initiate autophagy-related signaling.

As a protein kinase, mTOR is a key factor in the inhibition of autophagy as well as the regulation of cell growth and activity. mTOR directly phosphorylates ULK1 and various autophagy proteins to mediate the inhibition of autophagy. Conversely, autophagy is actively promoted under conditions in which mTOR activity is inhibited.

ATG family genes are Autophagy-Related Genes composed of more than 30 key genes involved in the initiation and progression of autophagy. These genes are necessary for the entire process of autophagy, including the initiation of autophagy, the formation of the autophagy membrane, the recognition of the substrate to be degraded, and the fusion reaction between the autophagy membrane and the lysosome.

DDIT4, also known as REDD1, is a protein that inhibits mTOR through the TSC protein. Therefore, an increase in DDIT4 is a gene that increases the activity of autophagy by inducing inhibition of mTOR.

In addition, according to an embodiment of the present invention, the pharmaceutical composition of the present invention has the effect of inhibiting the growth of cancer cells and the effect of inhibiting the growth of tumors in vivo. Therefore, the pharmaceutical composition of the present invention is cancer, more specifically melanoma, blood cancer, skin cancer, colorectal cancer, brain cancer, ovarian cancer, bladder cancer, breast cancer, uterine cancer, duodenal cancer, fibrosarcoma, kidney cancer, liver cancer, lung cancer , pancreatic cancer, prostate cancer, stomach cancer, bone tumor, endometrial cancer, etc. have anticancer effects.

In one embodiment of the present invention, the composition will additionally include an anticancer agent.

In one embodiment of the present invention, the anticancer agent is fluorouracil (Fluorouracil), irinotecan (Irinotecan), anti-PD1 antibody, bevacizumab, capecitabine (Capecitabine), cetuximab (Cetuximab), Ramucirumab, Oxaliplatin, Ipilimumab, Pembrolizumab, Leucovorin, Trifluritin/Tipiracil, Nivolumab, Nivolumab It is selected from the group consisting of panitumumab, regorafenib, aflibercept, and combinations thereof.

In one embodiment of the present invention, the anticancer agent is selected from the group consisting of fluorouracil (Fluorouracil), irinotecan (Irinotecan), anti-PD1 antibody, and combinations thereof.

In one embodiment of the present invention, the composition comprises 0.5 to 6 μM of lomitapide.

According to an embodiment of the present invention, the lomitapide may produce a synergistic anticancer effect against colon cancer cells HCT116 at 0.5 to 6 μM and the fluorouracil at 1 to 12 μM, and more specifically, the lomitapide shows the best effect on colon cancer cells HCT116 at 0.5 to 1.25 μM and fluorouracil at 1.25 to 5 μM.

According to an embodiment of the present invention, the lomitapide is 1.25 to 6 µM and the irinotecan is 1 to 12 µM to produce a synergistic anticancer effect against colon cancer cells HCT116, and more specifically, the lomitapide is 2.5 to 6 µM. to 6 μM and irinotecan exhibits the best effect on colorectal cancer cells HCT116 at 1.25 to 5 μM.

According to an embodiment of the present invention, the lomipatide may produce a synergistic anticancer effect against colorectal cancer cells HT29 at 0.5 to 1.25 μM and the fluorouracil at 10 to 22 μM.

According to an embodiment of the present invention, the lomipatide may exert an anticancer synergistic effect on colorectal cancer cells HT29 at 0.5 to 2.75 μM and the irinotecan at 4 to 22 μM, and more specifically, the lomitapide at 0.625 to 2.5 μM. μM and the irinotecan show the best effect on colorectal cancer cells HT29 at 5 to 20 μM.

According to an embodiment of the present invention, the lomitapide at 2-3 μM and the fluorouracil at 12.5 to 110 μM can produce an anticancer synergistic effect against colorectal cancer cells SW480.

According to an embodiment of the present invention, the lomitapide can produce a synergistic anticancer effect against colorectal cancer cells SW480 at 2 to 3 μM and the irinotecan at 0.6 to 60 μM, and more specifically, the lomitapide is 2 to 3 μM. 3 μM and the irinotecan show the best effect on colorectal cancer cells SW480 at 0.6 to 1.56 μM or 6.25 to 60 μM.

PD-1 is a major immune checkpoint receptor expressed by activated T and B cells and mediates immunosuppression. PD-1 is a member of the CD28 family of receptors, including CD28, CTLA-4, ICOS, PD-1, and BTLA. Two cell surface glycoprotein ligands for PD-1 have been identified: programmed death ligand-1 (PD-L1) and programmed death ligand-2 (PD-L2), which contain antigen-presenting cells as well as many It is expressed on human cancers and has been shown to down-regulate cytokine secretion and T cell activation upon binding to PD-1. Inhibition of the PD-1/PD-L1 interaction mediates potent antitumor activity in preclinical models.

According to another aspect of the present invention, the present invention provides a method for preventing or preventing cancer comprising administering to a subject a pharmaceutical composition comprising the above-described mTOR signaling inhibitor of the present invention as an active ingredient. provide a treatment method.

As used herein, the term "administration" or "administer" refers to directly administering a therapeutically or prophylactically effective amount of a composition of the present invention to a subject (individual) suffering from, or likely to suffer from, the subject disease. It means that the same amount is formed in the body of

The "therapeutically effective amount" of the composition means an amount of the composition sufficient to provide a therapeutic or prophylactic effect to a subject to which the composition is administered, and includes a "prophylactically effective amount".

In addition, as used herein, the term "subject (subject)" is a mammal, including humans, mice, rats, guinea pigs, dogs, cats, horses, cattle, pigs, monkeys, chimpanzees, baboons and rhesus monkeys. . Most specifically, the subject of the present invention is a human.

In one embodiment of the present invention, the step of administering the pharmaceutical composition to the subject is to administer the composition and the anticancer agent in combination.

In one embodiment of the present invention, the anticancer agent is selected from the group consisting of fluorouracil, irinotecan, anti-PD1 antibody, and combinations thereof.

Since the method for preventing or treating cancer of the present invention includes administering the pharmaceutical composition according to an aspect of the present invention, the description thereof is omitted to avoid excessive redundancy of the present specification for overlapping content. .

The features and advantages of the present invention are summarized as follows:

The present invention provides a pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active ingredient.

The composition of the present invention can be used as an anticancer agent by inhibiting the growth of cancer cells and exhibiting the efficacy of killing cancer cells.

1 shows the chemical formula of lomitapide.
Figure 2 shows the results of analyzing the shape of the cells after treating the normal human intestinal epithelial cells and colon cancer cell line HCT116 with DMSO control drug and lomitapide, respectively.
Figure 3 shows the results of analyzing the cell viability after treating the normal human intestinal epithelial cells and colon cancer cell line HCT116 with DMSO control drug and lomitapide, respectively.
4 shows the results of analysis of cell viability after treatment with DMSO control drug and lomitapide for three representative colorectal cancer cell lines (HCT116, HT29, SW480).
Figure 5a shows the results of analyzing the cell viability after lomitapide treatment for brain cancer cell lines.
Figure 5b shows the results of analyzing the viability of cells after treatment with lomitapide targeting breast cancer cell lines.
Figure 5c shows the results of analyzing the viability of the cells after lomitapide treatment on a blood cancer cell line.
Figure 5d shows the results of analyzing the cell viability after treatment with lomitapide targeting colon cancer cell lines.
Figure 5e shows the results of analyzing the viability of cells after treatment with lomitapide targeting lung cancer cell lines.
Figure 5f shows the results of analyzing the viability of the ovarian cancer cell line after treatment with lomitapide.
5g shows the results of analyzing the cell viability of gastric cancer cell lines after treatment with lomitapide.
Figure 5h shows the results of analyzing the survival rate of cells after treatment with lomitapide in bladder cancer, bone cancer, duodenal cancer, endometrial cancer, fibrosarcoma, kidney cancer, liver cancer, pancreatic cancer, prostate cancer, skin cancer, and melanoma cell lines. indicates.
6 shows the results of analyzing proteins related to emtor signaling in cells after treatment with a DMSO control drug and lomitapide for colon cancer cells HCT116 through Western blot.
Figure 7a shows the results of analysis of proteins related to emtor signaling and autophagy in the colon cancer cells SW480 after treatment with DMSO control drug and lomitapide through Western blot.
Figure 7b shows the results of analysis of proteins related to emtor signaling and autophagy in cells after treatment with DMSO control drug and lomitapide for colorectal cancer cells HT29 through western blot.
Figure 7c shows the results of analyzing the proteins related to emtor signaling and autophagy in the cells after treatment with DMSO control drug and lomitapide for breast cancer cells MDA-MB-231 through Western blot.
Figure 7d shows the results of analyzing the proteins related to emtor signaling and autophagy in the cells after treatment with DMSO control drug and lomitapide for breast cancer cells MDA-MB-468 through Western blot.
FIG. 7e shows the results of analyzing proteins related to emtor signaling and autophagy in gastric cancer cells after treatment with DMSO control drug and lomitapide for HS746T gastric cancer cells through Western blot.
FIG. 7f shows the results of analyzing proteins related to emtor signaling and autophagy in gastric cancer cells after treatment with DMSO control drug and lomitapide for SNU1 gastric cancer cells through Western blot.
Figure 7g shows the results of analyzing the proteins related to emtor signaling and autophagy in the cells after treatment with DMSO control drug and lomitapide for gastric cancer cells SNU216 through Western blot.
8 shows the results of analyzing the protein related to autophagy of cells after treatment with DMSO control drug and lomitapide for colon cancer cells HCT116 through Western blot.
Figure 9 shows the results of analysis of apoptosis by autophagy after treating the human colorectal cancer cell line HCT116 with DMSO control drug, lomitapide, and 3-methylamine (3-MA), respectively, through Western blot.
10 shows the results of analysis of apoptosis by autophagy after the human colon cancer cell line HCT116 was treated with a DMSO control drug, lomitapide, and 3-methylamine (3-MA), respectively, through a microscope.
11 shows the results of analyzing the significant gene-mechanism relationship using RNA-seq analysis after treating the human colon cancer cell line HCT116 with DMSO control drug and lomitapide, respectively.
12 shows the results of analyzing significant genes related to the autophagy mechanism using RNA-seq analysis after treating the human colon cancer cell line HCT116 with a DMSO control drug and lomitapide, respectively.
13 shows the results of measuring the degree of cancer cell colony proliferation after treatment with a DMSO control drug and lomitapide for colorectal cancer cells HCT116.
Figure 14a shows the results of comparing and analyzing the change in tumor size with the control group after treatment with lomitapide (50 mg/Kg) of colorectal cancer cells HCT116 xenografted into mice.
Figure 14b shows the results of analyzing the change in tumor size after treatment with lomitapide (25, 50 mg/Kg) of colorectal cancer cells HCT116 xenografted into mice.
Figure 14c shows the results of analyzing the change in tumor size after treatment with lomitapide (10 mg/Kg) of colon cancer cells xenografted to mice HCT116.
Figure 15a shows the results of analyzing the drug synergistic efficacy of 5-FU or irinotecan and lomitapide on colon cancer cells HCT116 cells.
Figure 15b shows the results of analyzing the drug synergistic efficacy of 5-FU or irinotecan and lomitapide on colon cancer cells HCT116 cells.
Figure 15c shows the results of analyzing the drug synergistic efficacy of 5-FU or irinotecan and lomitapide on colon cancer cells HCT116 cells.
Figure 16a shows the results of analyzing the drug synergistic efficacy of 5-FU or irinotecan and lomitapide in colorectal cancer cells HT29 cells.
Figure 16b shows the results of analyzing the drug synergistic efficacy of 5-FU or irinotecan and lomitapide in colorectal cancer cells HT29 cells.
Figure 16c shows the results of analyzing the drug synergistic efficacy of 5-FU or irinotecan and lomitapide in colorectal cancer cells HT29 cells.
Figure 17a shows the results of analyzing the drug synergistic efficacy of 5-FU or irinotecan and lomitapide in colorectal cancer cells SW480 cells.
Figure 17b shows the results of analyzing the drug synergistic efficacy of 5-FU or irinotecan and lomitapide in colorectal cancer cells SW480 cells.
Figure 17c shows the results of analyzing the drug synergistic efficacy of 5-FU or irinotecan and lomitapide in colorectal cancer cells SW480 cells.
Figure 18a shows the results of visual analysis of the synergistic effect after treatment with lomitapide and Anti-PD1 combination of colon cancer cells transplanted into mice MC38.
Figure 18b shows the results of analyzing the synergistic effect by measuring the tumor volume after lomitapide and Anti-PD1 combined treatment with colorectal cancer cells transplanted into mice MC38.
Figure 18c shows the synergistic effect of immune cells (CD4+, CD4+/CD44+, CD8+, CD8+/IFNγ, Foxp3, NK, NK/IFNγ) analysis of colon cells transplanted into mice MC38 after combined treatment with lomitapide and Anti-PD1 shows the confirmed results.
Figure 19a shows the results of visual analysis of synergistic effects after treatment with lomitapide and Anti-PD1 in combination with skin melanoma cells B16F10 transplanted into mice.
Figure 19b shows the results of analyzing the synergistic effect by measuring the tumor volume after lomitapide and Anti-PD1 combination treatment with skin melanoma cells transplanted into mice B16F10.
20 shows the results of analyzing the viability of organoids derived from colorectal cancer patients treated with lomitapide.

Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention in more detail, and it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples according to the gist of the present invention. .

Example

Throughout this specification, "%" used to indicate the concentration of a specific substance is (weight/weight) % for solid/solid, (weight/volume) % for solid/liquid, and Liquid/liquid is (volume/volume) %.

Example 1: Cell culture method

NCM460 cells (human colon normal cells), HCT116 cells (human colon cancer cells, p53 wild-type), HT29 cells (human colon cancer cells, p53 mutant), SW480 cells (human colon cancer cells, p53 mutant), MDA-MB-231 cells (human breast cancer cells), MDA-MB-468 cells (human breast cancer cells), HS746T cells (human gastric cancer cells), SNU1 cells (human gastric cancer cells), SNU2 cells (human gastric cancer cells), MC38 cells (murine colorectal cancer cells) , B16F10 cells (murine melanoma cells), were purchased from ATCC (American Type Culture Collection, Virginia, USA) and used.

NCM460 and HCT116 cells were cultured in McCoy's 5a medium (Sigma Aldrich, Missouri, USA) supplemented with 2 mM glutamine, 1% penicillin-streptomycin, and 10% fetal bovine serum (FBS) at 37 °C and 5% CO ₂ conditions. did. HT29 and SW480 cells were cultured in RPMI medium (Sigma Aldrich, Missouri, USA) supplemented with 2 mM glutamine, 1% penicillin-streptomycin, and 10% fetal bovine serum (FBS) at 37° C. and 5% CO ₂ conditions. . MDA-MB-231, MDA-MB-468, HS746T, MC38, B16F10 cells were cultured in DMEM medium supplemented with 2 mM glutamine, 1% penicillin-streptomycin and 10% fetal bovine serum (FBS) (Sigma Aldrich, Missouri, USA). ) at 37 ℃, 5% CO ₂ Incubated under conditions. SNU1, SNU2 cells were cultured in RPMI medium (Sigma Aldrich, Missouri, USA) supplemented with 2 mM glutamine, 1% penicillin-streptomycin, 25 mM HEPES, 25 mM NaHCO _{3 and 10% fetal bovine serum (FBS) at 37 °C,} It was cultured under the conditions of 5% CO _{2 .}

Example 2: Viability assay of cells treated with lomitapide

Example 2-1. Viability analysis of lomitapide-treated colon cancer cells

NCM460, HCT116, HT29, SW480 cells were seeded in 96-well plates at ^{a density of 10 4} cells/well, incubated at 37 °C for 24 hours, and then at various concentrations (0, 1, 2, 5, 10 μM) of Lomi It was treated with tapide (Sigma Aldrich, Missouri, USA). After lomitapide treatment Plates were incubated at 5% CO ₂ , 37° C. for 24 hours. Thereafter, 100 μL of assay reagent (CellTiter-Glo® Reagent) was added to each well, and luminescence was measured using a VICTOR X Multilabel Reader (PerkinElmer, Massachusetts, USA). %) was calculated. The results are shown in FIGS. 2 to 4 .

Through the above experiment, it was confirmed that in the case of the anticancer cells treated with lomitapide, the survival rate of the cells was decreased compared to that of the normal cells ( FIG. 2 ). The above results indicate that lomitapide has a cancer cell-specific anticancer effect.

In addition, through the above experiment, it was confirmed that the cell viability decreased as the dose of lomitapide increased in three representative colorectal cancer cell lines (HCT116, HT29, SW480) ( FIGS. 3 and 4 ).

The above results indicate that lomitapide is very effective in killing various colorectal cancer cells.

Example 2-2. Viability analysis of lomitapide-treated cancer cells

Cell viability was analyzed by treatment with various concentrations of lomitapide for 120 cancer cells other than colon cancer cells.

To analyze the viability of cells, cells were treated _{with lomitapide and incubated for 72 hours at 37 °C in 5% CO 2} or 10% CO ₂ conditions, equilibrated to room temperature for 1 hour, and CellTiterGlo reagent (Promega) was added. After 1 hour, the luminescence was measured, and the cell viability (%) was calculated therefrom. 120 types of cells were purchased from ATCC (American Type Culture Collection, Virginia, USA) and used.

The results of analyzing cell viability for various cancer cells are as follows (Table 1, FIGS. 5A to 5H).

division cell name IC50 (μM) target organ One A172 1.90 brain 2 A2058 1.40 skin 3 A2780 2.70 ovary 4 A375 1.80 skin 5 A498 2.90 kidney 6 A549 3.60 lung 7 AsPC-1 3.60 pancreas 8 BEN 4.20 lung 9 BT-20 5.00 breast 10 BxPC-3 5.70 pancreas 11 Caki-1 3.60 kidney 12 Caki-2 2.80 kidney 13 Colo 205 3.40 colon 14 COR-L279 2.70 lung 15 COV434 3.10 ovary 16 DLD-1 4.50 colon 17 DU-145 5.00 prostate 18 DV90 5.20 lung 19 EFM-19 3.60 breast 20 EFM-192A 4.00 breast 21 EFO-27 4.00 ovary 22 EPLC-272H 3.10 lung 23 H1299 4.80 lung 24 H2228 5.30 lung 25 H4 4.60 brain 26 H460 3.80 lung 27 HCC 1569 2.90 breast 28 HCC38 6.00 breast 29 HCC827 4.10 lung 30 HCT116 5.00 colon 31 HCT-15 3.50 colon 32 HEC-1-A 4.90 endometrium 33 HEC-1-B 6.90 endometrium 34 Hep3B2.1-7 2.70 liver 35 HL-60 3.30 blood 36 Hs 746T 2.50 stomach 37 HT-1080 3.90 fibrosarcoma 38 HT-29 1.60 colon 39 Hutu 80 3.00 duodenum 40 Ishikawa 3.60 endometrium 41 J82 3.60 bladder 42 JIMT-1 3.30 breast 43 Jurkat 3.00 blood 44 JVM-3 4.00 blood 45 K562 2.10 blood 46 KARPAS 299 1.40 blood 47 Kato III 5.40 stomach 48 KG-1 5.70 blood 49 KMS-12-BM 3.50 blood 50 LN229 2.70 brain 51 LnCap 3.50 prostate 52 LOU-NH91 5.70 lung 53 LOVO 3.40 colon 54 LP-1 5.40 blood 55 M07e 4.20 blood 56 MCAS 4.50 ovary 57 MCF-7 2.80 breast 58 MDA MB 231 3.20 breast 59 MDA MB 435 3.00 skin 60 MEC-1 3.30 blood 61 Mia PaCA 2 2.90 pancreas 62 MKN-1 3.70 stomach 63 MKN-45 3.90 stomach 64 MOLM-13 6.30 blood 65 MOLT-4 3.60 blood 66 MV4-11 6.20 blood 67 NCI-H1048 5.50 lung 68 NCI-H1437 3.10 lung 69 NCI-H1563 5.30 lung 70 NCI-H1573 4.70 lung 71 NCI-H1581 3.70 lung 72 NCI-H1703 5.70 lung 73 NCI-H1838 4.00 lung 74 NCI-H2009 3.30 lung 75 NCI-H2110 3.30 lung 76 NCI-H2286 3.40 lung 77 NCI-H292 3.00 lung 78 NCI-H441 3.20 lung 79 NCI-H82 3.20 lung 80 NCI-H838 3.80 lung 81 NCI-N87 3.30 stomach 82 OCI-AML3 2.10 blood 83 OCI-AML5 2.70 blood 84 OCI-LY19 2.80 blood 85 OPM-2 4.40 blood 86 OV56 4.80 ovary 87 OVCAR-3 5.30 ovary 88 OVK18 3.30 ovary 89 P31/FUJ 3.10 blood 90 PANC-1 4.50 pancreas 91 PC3 4.30 prostate 92 RERF-LC-Ad2 2.60 lung 93 RERF-LC-MS 3.20 lung 94 RKO 3.30 colon 95 RL95-2 3.20 endometrium 96 RPMI 8226 2.10 blood 97 Saos-2 EC 3.60 bone 98 SCLC-21H 3.00 lung 99 SJSA-1 3.10 bone 100 SK.BR-3 3.90 breast 101 SK-ES-1 3.50 bone 102 SK-LU-1 4.60 lung 103 SK-MEL-3 1.60 skin 104 SK-N-FI 3.40 brain 105 SK-N-MC 2.40 brain 106 SK-N-SH 3.20 brain 107 SK-OV3 3.30 ovary 108 SNU-1 2.80 stomach 109 SNU-216 3.10 stomach 110 SNU840 2.30 ovary 111 SW480 2.50 colon 112 SW620 3.60 colon 113 SW948 4.50 colon 114 T84 3.20 colon 115 T98G 3.80 brain 116 U118MG 2.50 brain 117 U251MG 5.40 brain 118 U2OS 2.80 bone 119 U87MG 3.10 brain 120 U-937 2.30 blood

Through the above experiment, it was confirmed that lomitapide produced anticancer effects at very low concentrations in various carcinomas and cancer cell lines, and the types were melanoma, blood cancer, skin cancer, colorectal cancer, brain cancer, ovarian cancer, bladder cancer, breast cancer, uterine cancer, These include duodenal cancer, fibrosarcoma, kidney cancer, liver cancer, lung cancer, pancreatic cancer, prostate cancer, gastric cancer, bone tumor, endometrial cancer, and the like.

Example 3: Cell signaling assay of lomitapide-treated cells

Example 3-1. Signal transduction analysis of lomitapide-treated colon cancer cells and breast cancer cells

In order to confirm the function of lomitapide in HCT116, SW480, HT29, MDA-MB-231, and MDA-MB-468 cell lines, the signaling effect of cells when lomitapide was added was examined.

Lomitapide-treated at various concentrations (0, 5, 10 μM) Cells were lysed with RIPA buffer containing a protease-inhibitor cocktail. Whole-cell lysate was incubated on ice for 30 minutes, then centrifuged at 4 °C, 13,300xg for 15 minutes, and the supernatant was collected. As a control, HCT116, SW480, HT29, MDA-MB-231, and MDA-MB-468 cells treated with 1 μM of Torin, known as an mTOR inhibitory component, were used.

For Western blot analysis, the supernatant obtained above was loaded on a 10% SDS-PAGE gel to separate, and the separated protein was blotted on a PVDF membrane. Then, the antibody was added to the blot as follows (Table 2), and incubated at 4° C. for 12 hours.

cell antibody HCT116 anti-p-AKT, anti-p-mTOR, anti-p-S6K, anti-p-S6, anti-p-ERK, anti-AKT, anti-S6K, anti-S6 (Cell Signaling Technology, Massachusetts, USA) , anti-alpha-tublin antibody (Sigma Aldrich, Missouri, USA) SW480 Anti-p-S6K, anti-p-S6, anti-S6K, anti-S6, anti-LC3 (Cell Signaling Technology), anti-alpha-tublin antibody (Sigma Aldrich) HT29 MDA-MB-231 MDA-MB-468

Then, the blot was washed with TBST (a mixture of tris-buffered saline (TBS) and Tween 20), and horseradish peroxidase-conjugated secondary antibody (Cell signaling) and 1 for time After incubation and washing, enhanced chemiluminescence (ECL; Amersham) was detected.

Through the above experiment, it was confirmed that lomitapide significantly reduced phosphorylation levels of target proteins (p-AKT, p-mTOR, p-S6K, p-S6) in a concentration-dependent manner in HCT116 ( FIG. 6 ). In addition, it was confirmed that lomitapide significantly reduced phosphorylation levels of target proteins (p-S6K, p-S6) in SW480, HT29, MDA-MB-231, and MDA-MB-468. It was confirmed that the induction of LC3I to LC3II was increased ( FIGS. 7A to 7D ). These results indicate that lomitapide is very effective in inhibiting emtor (mTOR) and the upper signaling pathways of emtor (AKT, p-S6K, p-S6).

Example 3-2. Signal transduction analysis of lomitapide-treated gastric cancer cells

In order to confirm the function of lomitapide in HS746T, SNU1, and SNU2 cell lines, the signaling effect of cells when lomitapide was added was examined.

Lomitapide-treated at various concentrations (0, 5, 10 μM) Cells were lysed with RIPA buffer containing a protease-inhibitor cocktail. Whole-cell lysate was incubated on ice for 30 minutes, then centrifuged at 4 °C, 13,300xg for 15 minutes, and the supernatant was collected. As a control, 1 μM of Torin, known as an mTOR inhibitory component, was treated. cells were used.

For Western blot analysis, the supernatant obtained above was separated by loading on a 10% SDS-PAGE gel, and the separated protein was blotted on a PVDF membrane. Then, the antibody was added to the blot as follows (Table 3), and incubated at 4° C. for 12 hours.

cell antibody HS746T Anti-p-S6K, anti-p-S6, anti-S6K, anti-S6, anti-LC3 (Cell Signaling Technology), anti-alpha-tublin antibody (Sigma Aldrich) SNU1 SNU2

Then, the blot was washed with TBST (a mixture of tris-buffered saline (TBS) and Tween 20), and horseradish peroxidase-conjugated secondary antibody (Cell signaling) and 1 for time After incubation and washing, enhanced chemiluminescence (ECL; Amersham) was detected.

Through the above experiment, it was confirmed that lomitapide significantly reduced the phosphorylation level of p-S6K, p-S6) of target proteins in a concentration-dependent manner, and it was confirmed that lomitapide increased the induction of LC3I to LC3II. (FIGS. 7E-7G). These results indicate that lomitapide is very effective in inhibiting emtor (mTOR) and the upper signaling pathways of emtor (AKT, p-S6K, p-S6).

Example 4: Autophagy assay of cells treated with lomitapide

In order to confirm the correlation between the cellular autophagy function of lomitapide in the HCT116 cell line, the signaling effect of cells when lomitapide was added was examined.

Lomitapide-treated at various concentrations (0, 5, 10 μM) for analysis of changes in cell signaling involved in cell autophagy HCT116 cells were analyzed by Western blot. For Western blotting, the supernatant obtained in Example 3 was loaded on a 10% SDS-PAGE gel to separate, and the separated protein was blotted on a PVDF membrane. Then, anti-p-AMPK, anti-LC3, and anti-p-ULK1 antibodies (Cell Signaling Technology, Massachusetts, USA) were added to the blot, and incubated at 4° C. for 12 hours. Then, the blot was washed with TBST (a mixture of tris-buffered saline (TBS) and Tween 20), and horseradish peroxidase-conjugated secondary antibody (Cell signaling) and 1 for time After incubation and washing, enhanced chemiluminescence (ECL; Amersham) was detected.

Through the above experiment, it was confirmed that lomitapide significantly increased the phosphorylation level of AMPK, decreased the phosphorylation level of ULK1, and significantly increased the induction of LC3I to LC3II in a concentration-dependent manner ( FIG. 8 ). These results indicate that lomitapide has the effect of greatly increasing the expression of proteins involved in the autophagy mechanism.

In addition, in the case of simultaneous treatment of lomitapide and 3-methylamine (3-MA, Sigma Aldrich, Missouri, USA), which is an autophagy function inhibitor, in order to confirm the correlation between the cellular autophagy function of lomitapide in the HCT116 cell line. The signaling effect of the cells and the viability of the cells were examined.

Lomitapide-treated at 5 μM for analysis of changes in cell signaling involved in cell autophagy HCT116 cells and HCT116 cells treated with 1-mM 3-MA and 5 μM of lomitapide were analyzed by Western blot. The Western blotting procedure is the same as the Western blotting procedure described above except that anti-LC3 was used as the antibody.

In addition, for cell viability analysis, HCT116 cells were inoculated into a 96-well plate at ^{a density of 10 4} cells/well, and after incubation at 37°C for 24 hours, 5 μM of lomitapide, 3-MA 1 mM or 5 It was treated with μM lomitapide and 1-mM 3-MA. after processing Plates were incubated at 5% CO ₂ , 37° C. for 24 hours. Thereafter, 100 μL of an assay reagent (CellTiter-Glo® Reagent) was added to each well and the results were observed under a microscope.

Through the above experiment, it was confirmed that HCT116 cells treated with lomitapide and 3-MA significantly reduced the induction of LC3I to LC3II ( FIG. 9 ). In addition, it was confirmed that cell death was inhibited in the case of HCT116 cells treated with lomitapide and 3-MA through cell viability analysis (FIG. 10).

The above results indicate that lomitapide of the present invention inhibits AMPK and mTOR signaling processes, thereby increasing the expression of cellular autophagy mechanism proteins.

Example 5: Analysis of significant gene expression and mechanism of lomitapide-treated cells (RNA-seq assay)

Gene set enrichment analysis (GSEA) and Pathway enrichment analysis were performed to analyze genes significantly expressed through RNA-seq experiments and related mechanisms to confirm the relationship between the drug mechanism of lomitapide and genes. was carried out through

GSEA is an analysis method for extracting a significant gene-set in which expression values of two classes show a statistically significant difference from among various gene-sets constructed based on biological characteristics.

In order to finally detect significant gene-sets, genes with specific functions among all genes used in RNA-seq experiments using a gene annotation database (KEGG pathway, Gene Ontology, etc.) with various biological information about genes were identified. Various gene-sets were discovered by grouping, and significant genes were determined by referring to the difference in expression values between two classes within each gene-set, and statistically significant gene-sets were finally detected based on this.

As for the mechanisms to which a significant gene set belongs, cancer-related mechanisms related to the Emtor mechanism including autophagy were detected, and in addition, immune, aging, and diabetes-related mechanisms were detected ( FIG. 11 , Table 4).

division gene function GeneRatio associated disease One MAPK signaling pathway 0.31 cancer 2 Kaposi sarcoma-associated herpesvirus infection 0.4 cancer 3 Viral carcinogenesis 0.42 cancer 4 breast cancer 0.37 breast cancer 5 FoxO signaling pathway 0.52 cancer 6 Apoptosis 0.35 cancer 7 TNF signaling pathway 0.62 Inflammation 8 osteoclast differentiation 0.81 osteoporosis 9 IL-17 signaling pathway 0.45 inflammation, cancer 10 colorectal cancer 0.58 colorectal cancer 11 Transcriptional misregulation in cancer 0.4 cancer 12 Hepatitis B 0.33 inflammation, cancer 13 Non-alcoholic fatty liver disease (NAFLD) 0.33 Nonalcoholic hepatitis, cirrhosis and liver failure 14 Parathyroid hormone synthesis, secretion and action 0.53 Calcium metabolism, cancer 15 Adrenergic signaling in cardiomyocytes 0.62 heart disease 16 Epithelial cell signaling in Helicobacter pylori infection 0.38 gastritis, stomach cancer 17 Human T-cell leukemia virus 1 infection 0.35 leukemia 18 Cytokine-cytokine receptor interaction 0.37 inflammation, cancer 19 Autophagy - animal 0.6 Cancer, metabolic disease 20 C-type lectin receptor signaling pathway 0.16 immunity, cancer 21 Spliceosome 0.88 - 22 Relaxin signaling pathway 0.6 heart disease 23 AGE-RAGE signaling pathway in diabetic complications 0.53 diabetic complications 24 Pertussis 0.33 whooping cough 25 PI3K-Akt signaling pathway 0.24 cancer 26 Fluid shear stress and atherosclerosis 0.31 arteriosclerosis 27 ErbB signaling pathway 0.69 cancer 28 NF-kappa B signaling pathway 0.46 inflammation, cancer 29 Pathways in cancer 0.3 cancer 30 Neuroactive ligand-receptor interaction 0.52 - 31 Endocrine resistance 0.41 metabolic disease 32 Signaling pathways regulating pluripotency of stem cells 0.39 cancer 33 Peroxisome 0.83 - 34 Protein processing in endoplasmic reticulum 0.26 - 35 Shigellosis 0.38 bacterial dysentery

In addition, it was found that autophagy-related genes, such as ATG family, ULK1, and DDIT4, were significantly expressed through Differentially Expressed Genes analysis (DEG analysis) related to the autophagy mechanism (FIG. 12). DEG analysis is an analysis that selects a candidate group for a significant gene (Differentially Expressed Genes) with a difference in expression between the control group and the control group by measuring and statistically processing the expression value of the gene.

As can be seen from the above results, the effect of lomitapide on cancer cells is shown through i) inhibition of the emtor signaling mechanism and ii) gene expression related to autophagy.

Example 6: Cell colony forming assay of lomitapide-treated cells

In order to confirm the correlation between lomitapide and cancer cell colony growth rate in HCT116 cell line, the cancer cell colony growth rate was examined when lomitapide was added to the wells culturing HCT116 cells.

HCT116 cells were seeded in a 12-well plate at ^{a density of 10 5} cells/well, and after incubation at 37° C. for 24 hours, the cells were treated with 0, 5 μM lomitapide. After lomitapide treatment Plates were incubated at 5% CO ₂ , 37° C. for 48 hours. After that, 500 μL of crystal violet was added to each well, and the cells were stained at room temperature for 10 minutes to analyze the proliferation of the cells.

Through the experiment, it was confirmed that the cell proliferation rate in the wells treated with lomitapide was significantly lower than that in the wells not treated with lomitapide ( FIG. 13 ).

These results indicate that lomitapide has a very good anticancer effect.

Example 7: Analysis of the anticancer effect of lomitapide confirmed through animal experiments

In order to confirm the anticancer effect of lomitapide in the mouse xenograft model, the size of the tumor was examined after lomitapide was treated in the tumor-transplanted mice.

Animal experiments were performed according to guidelines approved by the Animal Experimental Ethics Committee of the Korea Advanced Institute of Science and Technology. HCT116 cells (4x10 ⁶ ) were implanted by subcutaneous injection into male BALB/c nude mice aged 8-12 weeks. After the mean tumor volume ^{reached 50 mm 3} , mice were randomly assigned to two different groups (5 mice/group). The mouse body weight and tumor diameter were measured once every two days. Tumor volume was evaluated according to the general formula 0.5x(width) ² x(Length) using calipers, and Student's T test was used to determine P-values. For lomitapide treatment, 1, 2, 3, 7, 8, 9, 13, 14, 15, and 16 days after the start of the experiment, 50 mg/kg of lomitapide was administered to the mouse. Injected into the abdominal cavity. As a control, DMSO was injected intraperitoneally into mice ( FIG. 14A ).

In addition, additional animal experiments were performed, and 25, 50 mg/kg of lomitapide was injected into the abdominal cavity of mice over a total of 5 times at intervals of 2 days after the start of the experiment. As a control, DMSO was injected intraperitoneally into mice ( FIG. 14b ).

In addition, additional animal experiments were performed, and 10 mg/kg of lomitapide was intratumoral injection of 10 mg/kg of lomitapide for a total of 5 times at an interval of 2 days after the start of the experiment. As a control, DMSO was injected intratumorally in mice ( FIG. 14c ).

Through the experiment, it was confirmed that the growth of the tumor in the lomitapide-treated mouse xenograft model was inhibited when compared to the control group (FIG. 14).

These results indicate that lomitapide has a very good anticancer effect.

Example 8: Analysis of synergistic effects following anticancer drug combination treatment (Drug synergy)

HCT116, HT29, SW480 cells were seeded in 96-well plates and treated without drug or with single drugs or in combination at the indicated concentrations for 48 hours. The final concentration of DMSO in each well was <0.01%, and it was confirmed that the treated amount had no observable toxicity in cells, and the following drugs were used for analysis.

drug name Manufacturer and Cat num. Lomitapide Sellekchem, S7635 5-FU Sigma Aldrich, F6627-1G Irinotecan Sellekchem, S2217

HCT116 cells were seeded at 10,000 cells/well, and HT29 and SW480 cells were seeded at 5,000 cells/well. After 48 hours of incubation, 100 μL of assay reagent (CellTiter-Glo® Reagent) was added to each well and luminescence was measured using a VICTOR X Multilabel Reader (PerkinElmer, Massachusetts, USA). The percent survival was calculated.

Drug synergy was assessed using the HSA model. HSA scores were divided into the ranges of > 10, -10 to 10, and < -10, indicating synergy, additive and antagonistic effects, respectively. Scores were expressed as individual scores in 4 x 4, 3 x 3, 2 x 7 matrices of drug dose, and the distribution of the overall drug response effect was shown in 2D and 3D contour graphs.

Through the above experiment, it was confirmed that synergistic effect appeared when lomitapide was treated in combination with two drugs used as conventional colorectal cancer drugs, that is, 5-FU or irinotecan ( FIGS. 15 to 17 ).

Example 9: Analysis of synergistic effects according to combination treatment of lomitapide and Anti-PD1 through animal experiments

For the allograft tumor mouse model, two types of MC38 colorectal cancer and B16F10 skin melanoma cell lines were used in female C57B6/N mice (wild-type, 6 weeks old). MC38 colorectal cancer and B16F10 cutaneous melanoma cells were injected subcutaneously at ^{2 x 10 5 .} Lomitapide used in both models was formulated with 45% saline, 40% PEG300, 5% Tween-80, and 10% DMSO.

For MC38 colorectal cancer, lomitapide was administered intraperitoneally at a dose of 20 mg/kg from 10 days after tumor injection for 5 doses, and 10 mg/kg anti-PD-1 mAb (clone RMP1-14, BioXCell, West Lebanon, NH , USA) or a rat IgG2a isotype control (clone 2A3, BioXCell, BE0089) were administered in PBS on days 1, 4, 7, and 10 ( FIGS. 18A and 18B ).

In the case of B16F10 cutaneous melanoma, lomitapide was administered at a dose of 40 mg/kg three times intraperitoneally on days 3, 5, and 7 from 10 days after tumor injection, and 20 mg/kg anti-PD-1 mAb (clone RMP1-14, BioXCell, West Lebanon, NH, USA) or rat IgG2a isotype control (clone 2A3, BioXCell, BE0089) were administered on days 1, 3, 5, and 7 in PBS. Mice were immediately euthanized when signs of distress were observed, 20% weight loss of normal body weight, or tumor volume ^{exceeded 1000 mm 3} ( FIGS. 19A and 19B ).

Through the two experiments, it was confirmed that the tumor growth of the lomitapide-treated mouse allograft model was inhibited and decreased when compared to the control group, and compared with the experimental group treated with anti-PD-1 mAb. In this case, even more excellent growth inhibition and reduction were observed, and in the experimental group treated with lomitapide and anti-PD-1 mAb, a synergistic effect on tumor size reduction was confirmed.

In addition, according to the analysis of immune cells (CD4+, CD4+/CD44+, CD8+, CD8+/IFNγ, Foxp3, NK, NK/IFNγ) in tumor cells of MC38 colorectal cancer, lomitapide alone and anti-PD-1 mAb When comparing the treated experimental groups, the lomitapide alone treated group had higher (CD4+, NK, NK/IFNγ) or similar levels (CD4+/CD44+, CD8+, CD8+/IFNγ, Foxp3) than the experimental group treated with anti-PD-1 mAb. ) was confirmed, and it was confirmed that the expression of all immune cells was significantly increased in the experimental group treated with both drugs at the same time (FIG. 18c).

Example 10: Viability of organoids from colorectal cancer patients treated with lomitapide analysis

As a result of analyzing the viability of organoids by lomitapide treatment using two types of colon cancer patient-derived organoids (organoids 01-46 years old male, organoid 02-74 years old female), lomitapide It was confirmed that as the dose of the organoid increased, the survival rate of the organoid decreased (FIG. 20). The above results indicate that lomitapide is very effective in killing various colorectal cancer cells.

1. Imaging based evaluation of drugs

Organoids derived from colorectal cancer patients are cultured in 48 well plates (SPL, cat 32048) for 5-7 days.

Remove the organoids from the plate by pipetting using 200 μL of phosphate buffered saline (DPBS; Welgene LB001-02) and transfer to a new tube.

Hoechst (Thermo, #H-1399) was put into the culture medium at a ratio of 1:2,000 for organoids, and then _{reacted for 30 minutes at 37 ° C., 5% CO 2 in an} incubator for staining.

Stained organoids are centrifuged for 1,350 rpm/3 min to remove the supernatant.

After putting the same amount of organoids in a 96-well plate, add the same volume of drugs.

After dispensing, the number, state, and area of organoids are checked through DAPI signals using Cytation5 (Biotek), a high-contents imaging-based screening equipment.

Thereafter, the change of the organoid area was observed for a total of 3 times/72 hours every 24 hours without changing the culture medium.

Based on the raw data derived from the Cytation5 equipment, the following formula was applied to calculate the efficacy of the drug. In addition, the overall efficacy of a drug at a specific concentration is defined as the sum of organoid growth inhibition and organoid death.

^{The area (μm 2} ) of each organoid stained with Hoechst was confirmed using Cytation5, and the area of all organoids in each well was summed.

The difference between the area of the organoid after drug treatment for 72 hours and the area of the organoid at the first 0 hour was calculated as a value.

2. Evaluation calculation formula

To observe organoid death due to a specific concentration of drug, the size change due to organoid death was calculated. Formula 1).

The overall efficacy evaluation can be calculated as the ratio of damaged organoids in the drug-treated group to the ratio of damaged organoids in the untreated group after 72 hours of drug treatment (Formula 2).

In conclusion, the experiments described in the above Examples show that lomitapide inhibits emtor-related signal transduction and activates the autophagy mechanism of cells to produce an anticancer effect, and synergistic effect when lomitapide is administered in combination with an existing anticancer agent. indicates that it causes

Claims (10)

A pharmaceutical composition for preventing or treating cancer comprising an mTOR signaling inhibitor as an active ingredient.
The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the emtor signaling inhibitor is lomitapide, a pharmaceutically acceptable salt thereof, or an optical isomer thereof.
The pharmaceutical composition for preventing or treating cancer according to claim 2, wherein the lomitapide is represented by the following formula (I):
[Formula I]

The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is a solid cancer.
5. The method of claim 4, wherein the solid cancer is melanoma, brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, brain lymphoma, oligodendroglioma, ependymoma, brainstem tumor, head and neck tumor, laryngeal cancer, oropharyngeal cancer, nasal/sinus cancer , nasopharyngeal cancer, salivary gland cancer, hypopharyngeal cancer, thyroid cancer, oral cancer, chest tumor, small cell lung cancer, non-small cell lung cancer, thymus cancer, mediastinal tumor, esophageal cancer, breast cancer, male breast cancer, abdominal cancer, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, Pancreatic cancer, small intestine cancer, colorectal cancer, rectal cancer, anal cancer, bladder cancer, kidney cancer, male genital tumor, penile cancer, prostate cancer, female genital tumor, cervical cancer, endometrial cancer, ovarian cancer, uterine sarcoma, vaginal cancer, female external reproductive system Stage cancer, female urethral cancer, bone tumor, duodenal cancer, fibrosarcoma, and will be selected from the group consisting of skin cancer, a pharmaceutical composition for the prevention or treatment of cancer.
The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is a blood cancer.
7. The method of claim 6, wherein the hematologic cancer is selected from the group consisting of acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, chronic lymphocytic leukemia, acute monocytic leukemia, multiple myeloma, Hodgkin's lymphoma and non-Hodgkin's lymphoma. The pharmaceutical composition for the prevention or treatment of cancer.
The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the composition further comprises an anticancer agent.
The method of claim 8, wherein the anticancer agent is fluorouracil, irinotecan, anti-PD1 antibody, bevacizumab, capecitabine, cetuximab, ramucirumab (Ramucirumab), Oxaliplatin, Ipilimumab, Pembrolizumab, Leucovorin, Trifluritin/Tipiracil, Nivolumab, Panitumumab (Panitumumab), regorafenib (Regorafenib), aflibercept, and a pharmaceutical composition for the prevention or treatment of cancer, which is selected from the group consisting of a combination thereof.
The pharmaceutical composition for preventing or treating cancer according to claim 9, wherein the anticancer agent is selected from the group consisting of fluorouracil, irinotecan, anti-PD1 antibody, and combinations thereof.

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