WO2021241862A1 - Pharmaceutical composition for cancer prevention or treatment, comprising inositol polyphosphate multikinase inhibitor as active ingredient - Google Patents

Pharmaceutical composition for cancer prevention or treatment, comprising inositol polyphosphate multikinase inhibitor as active ingredient Download PDF

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WO2021241862A1
WO2021241862A1 PCT/KR2021/003295 KR2021003295W WO2021241862A1 WO 2021241862 A1 WO2021241862 A1 WO 2021241862A1 KR 2021003295 W KR2021003295 W KR 2021003295W WO 2021241862 A1 WO2021241862 A1 WO 2021241862A1
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cancer
pharmaceutical composition
tumor
vilazodone
preventing
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Korean (ko)
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김세윤
이보아
박승주
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한국과학기술원
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/496—Non-condensed piperazines containing further heterocyclic rings, e.g. rifampin, thiothixene or sparfloxacin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia

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  • the present invention was made under project number NRF-2018R1A2B2005913 supported by the Ministry of Science and ICT A study on the function of metabolic stress signal regulation by polyphosphate multikinase”, organized by the Korea Advanced Institute of Science and Technology, and the research period is from March 1, 2019 to February 29, 2020.
  • the present invention has been made under project number NRF-2018R1A5A1024261 under the support of the Ministry of Science and ICT.
  • the host institution is the Korea Advanced Institute of Science and Technology, and the research period is from 2019.03.01 to 2020.02.29.
  • the present invention relates to a pharmaceutical composition for preventing or treating cancer comprising an inositol polyphosphate multikinase inhibitor as an active ingredient.
  • 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. In recent years, the incidence of colorectal cancer has increased sharply with the westernization of diet. Its rate of increase continues to increase.
  • colorectal cancer can be divided into surgical resection, chemotherapy, and radiation therapy.
  • Polyps which are the previous stages of colorectal cancer, or early colon/rectal cancer limited to polyps, can be treated with polypectomy.
  • fluorescent anticancer drugs such as irinotecan, oxaliplatin, capecitabine, and TAS-102 and epithelial cell growth cetuximab, Panitu, as a targeted anticancer agent that targets epidermal growth factor receptor (EGFR), vascular endothelial growth factor (VEGF), or vascular endothelial growth factor receptor (VEGF-R)
  • EGFR epidermal growth factor receptor
  • VEGF vascular endothelial growth factor
  • VEGF-R vascular endothelial growth factor receptor
  • the present inventors made intensive research efforts to develop compounds for inhibiting cancer cell growth and killing cancer cells. As a result, in the case of a composition comprising an Inositol Polyphosphate Multikinase (IPMK) inhibitor as an active ingredient, it was found that it is effective in cancer treatment, thereby completing the present invention.
  • IPMK Inositol Polyphosphate Multikinase
  • an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising an IPMK inhibitor as an active ingredient.
  • the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an inositol polyphosphate multikinase inhibitor as an active ingredient.
  • Inositol polyphosphate is an inositol metabolite synthesized by sequential phosphorylation of inositol 1,4,5-trisphosphate (IP3) by a series of inositol kinases and biosynthesis, and is bound to the inositol ring structure It is called IP4, IP5, or IP6 depending on the number of phosphate groups.
  • IP7 and IP8 can be synthesized, and this type of inositol polyphosphate is specifically called inositol pyrophosphate.
  • IPMK Inositol polyphosphate multikinase
  • the IPMK inhibitor may refer to a substance that inhibits the activity of IPMK itself or inhibits the activity of an upper-level factor that increases the activity of IPMK.
  • the IPMK inhibitor has the effect of inhibiting the activity of IPMK, regulating the activation of the Akt signaling pathway, and inhibiting the growth of cancer cells through reduction of Akt 473 phosphorylation. Therefore, IPMK inhibitors may exhibit anticancer effects against various carcinomas.
  • the inositol polyphosphate multikinase inhibitor is vilazodone.
  • Villazodone (trade name: Viibryd) is a drug used as an antidepressant developed by Merck in Germany, and the US approved the use of vilazodone as an antidepressant in 2011.
  • vilazodone is 5-(4-[4-(5-cyano-1H-indole-3-yl)butyl]piperazin-1-yl)benzofuran-2-carboxamide (5-(4 -(4-(5-Cyano-1H-indol-3-yl)butyl)piperazin-1-yl)benzofuran-2-carboxamide).
  • the inositol polyphosphate multikinase inhibitor is vilazodone, a pharmaceutically acceptable salt thereof, or an optical isomer thereof.
  • the cancer is a solid cancer.
  • 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
  • the solid cancer is 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 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,
  • the cancer is a hematologic cancer.
  • 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.
  • the hematologic cancer is acute myelocytic 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 composition as an active ingredient.
  • 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; it is 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.
  • a lubricant e.g., a talc, a kaolin, a kaolin, a kaolin, a kaolin, kaolin, kaolin, kaolin, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, sorbitol, a talct, a talct, a talct, a stevia, glycerin, glycerin, glycerin,
  • 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 method, 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.
  • the daily dose of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg.
  • the term “pharmaceutically effective amount” refers to an amount sufficient to prevent or treat the above-mentioned diseases.
  • prevention refers to the prevention or protective treatment of a disease or disease state.
  • 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.
  • the dosage form can 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.
  • the pharmaceutical composition comprising vilazodone of the present invention has the effect of inhibiting the activity of IPMK and increasing the expression of proteins involved in the autophagy mechanism of cells.
  • 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 has an anticancer effect on cancer, more specifically colon cancer.
  • the present invention provides a method for preventing or treating cancer comprising administering to a subject a pharmaceutical composition comprising the above-described inositol polyphosphate multikinase inhibitor of the present invention as an active ingredient provides
  • 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 to be administered, and includes a “prophylactically effective amount”.
  • 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.
  • 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 present invention provides a pharmaceutical composition for preventing or treating cancer comprising an inositol polyphosphate multikinase inhibitor as an active ingredient.
  • 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.
  • Figure 1a shows the results of analyzing the inhibitory effect of inositol kinase activity of vilazodone through IP kinase assay.
  • Figure 1b shows the results of analyzing the metabolites of IPMK after treating NIH3T3-L1 fibroblasts with DMSO, vilazodone, and quercetin through HPLC.
  • Figure 1c shows the result of analyzing the phosphorylation level of Akt after treatment with DMSO drug and vilazodone for HCT116 cells through Western blot.
  • Figure 2a shows the results of analyzing the cell viability after treating the colon cancer cell line HCT116 with DMSO and vilazodone, respectively.
  • Figure 2b shows the results of analyzing the cell viability after treating the colon cancer cell line HT29 with DMSO and vilazodone, respectively.
  • Figure 2c shows the results of analyzing the cell viability after treating the colon cancer cell line SW480 with DMSO and vilazodone, respectively.
  • FIG. 3 shows the results of analysis of proteins related to autophagy of cells after treatment with DMSO and vilazodone for three representative colorectal cancer cell lines (HCT116, HT29, SW480) through Western blot.
  • % 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) %.
  • 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
  • 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.
  • 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. .
  • Example 2-1 IPMK inhibitory effect of vilazodone confirmed by performing IP kinase assay
  • the IPMK inhibitory effect of vilazodone was measured using the ADP-Glo Kinase kit (Promega, Wisconsin, USA).
  • kinase assay buffer 50 mM HEPES [pH 7.4], 10 mM MgCl2, 50 mM KCl), 50 ng recombinant IPMK, 10 ⁇ M ATP, and 100 ⁇ M vilazodone or quercetin (Sigma Aldrich, Missouri, USA) was added to the experiment. The reaction was quenched with ADP-Glo reagent at room temperature for 40 min. After that, kinase detection substrate was added and the reaction was incubated for an additional 30 minutes. Luminescence was detected by plate reading.
  • Example 2-2 IPMK inhibitory effect of vilazodone confirmed by HPLC
  • NIH3T3-L1 fibroblasts (2 ⁇ 10 5 cells) were inoculated in DMEM 60 mm medium supplemented with 10% calf serum. Cells were labeled with 60 ⁇ Ci [ 3 H]-myo-inositol for 3 days.
  • Villazodone and quercetin were dissolved in DMSO to a concentration of 0.1%, and 10 ⁇ M of vilazodone or quercetin dissolved in DMSO was treated for the experiment.
  • Soluble inositol metabolites were extracted with perchloric acid buffer, and the remaining insoluble inositol metabolites were extracted using 0.1 M NaOH and 0.1% Triton X-100. 3H-labeled IP was analyzed by high performance liquid chromatography (HPLC), and each fraction was measured using a liquid scintillation counter.
  • Example 2-3 IPMK inhibitory effect of vilazodone confirmed by immunoblotting
  • HCT116 cells were seeded in a 6-well plate at a density of 4 ⁇ 10 4 cells/well and treated with 10 ⁇ M of vilazodone one day later. After 4 h of vilazodone treatment, cells were washed with NP-40 buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM EDTA [pH 8.0], 50 mM NaF, 10 mM Na-Pi). lysate, and a cocktail of protein inhibitors [Roche, Switzerland]). The cell lysate was loaded on a 4 to 12% Bis-Tris SDS-PAGE gel, and transferred to a nitrocellulose membrane.
  • Phospho-S473 Akt, Total Akt (Cell Signaling Technology, Massachusetts, USA), and ⁇ -tubulin (Sigma Aldrich, Missouri, USA) antibodies were diluted 1:1,000 and used.
  • the secondary antibody was diluted 1:2,000 and used. Proteins were detected using a chemiluminescent immunoblot detection system (Bio-Rad, California USA).
  • 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, 1.25, 2.5, 5, 10, 20 ⁇ M) of Villa It was treated with crude pigs (Selleckchem, Texas, USA). After treatment with vilazodone Plates were incubated at 5% CO 2 , 37° C. for 24, 48, 72 hours. Thereafter, 100 ⁇ L of an assay reagent was added to each well of the cells, and luminescence was measured using a VICTOR X Multilabel Reader (PerkinElmer, Massachusetts, USA), and is shown in FIG. 2 .
  • TBST a mixture of tris-buffered saline (TBS) and Tween 20
  • Cell signaling horseradish peroxidase-conjugated secondary antibody
  • ECL enhanced chemiluminescence
  • vilazodone significantly decreased the phosphorylation level of ULK1 and significantly increased the induction of LC3I to LC3II in a concentration-dependent manner ( FIG. 3 ). These results indicate that vilazodone has the effect of significantly increasing the expression of proteins involved in the autophagy mechanism.
  • Example 5 Assay for colony growth of vilazodone-treated cells (cell colony forming assay)
  • the cancer cell colony growth rate was examined when vilazodone was added to the wells culturing HT29 cells.
  • HT29 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, 10, 20 ⁇ M vilazodone. After treatment with vilazodone Plates were incubated at 5% CO 2 , 37° C. for 48 hours. After that, 500 ⁇ L 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.

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Abstract

The present invention relates to a pharmaceutical composition for cancer prevention or treatment, comprising an inositol polyphosphate multikinase inhibitor as an active ingredient. The composition according to the present invention exhibits the effects of inhibiting the growth of cancer cells and killing cancer cells and thus can be used as an anticancer agent.

Description

이노시톨 다인산 멀티키나아제 억제제를 유효성분으로 포함하는 암의 예방 또는 치료용 약제학적 조성물Pharmaceutical composition for preventing or treating cancer comprising an inositol polyphosphate multikinase inhibitor as an active ingredient
본 발명은 과학기술정보통신부 지원 하에서 과제번호 NRF-2018R1A2B2005913에 의해 이루어진 것으로서, 상기 과제의 연구관리전문기관은 한국연구재단, 연구사업명은 “중견연구자지원사업”, 연구과제명은 “골수성 세포 특이적 이노시톨 다인산 멀티키나아제에 의한 대사적 스트레스 신호조절기능연구”, 주관기관은 한국과학기술원, 연구기간은 2019.03.01 ~ 2020.02.29 이다. The present invention was made under project number NRF-2018R1A2B2005913 supported by the Ministry of Science and ICT A study on the function of metabolic stress signal regulation by polyphosphate multikinase”, organized by the Korea Advanced Institute of Science and Technology, and the research period is from March 1, 2019 to February 29, 2020.
본 발명은 과학기술정보통신부 지원 하에서 과제번호 NRF-2018R1A5A1024261에 의해 이루어진 것으로서, 상기 과제의 연구관리전문기관은 한국연구재단, 연구사업명은 “선도연구과제”, 연구과제명은 “세포 정체성 연구 센터”, 주관기관은 한국과학기술원, 연구기간은 2019.03.01 ~ 2020.02.29 이다. The present invention has been made under project number NRF-2018R1A5A1024261 under the support of the Ministry of Science and ICT. The host institution is the Korea Advanced Institute of Science and Technology, and the research period is from 2019.03.01 to 2020.02.29.
본 특허출원은 2020년 5월 26일에 대한민국 특허청에 제출된 대한민국 특허출원 제10-2020-0063238호에 대하여 우선권을 주장하며, 상기 특허출원의 개시사항은 본 명세서에 참조로서 삽입된다. This patent application claims priority to Korean Patent Application No. 10-2020-0063238, filed with the Korean Intellectual Property Office on May 26, 2020, the disclosure of which is incorporated herein by reference.
본 발명은 이노시톨 다인산 멀티키나아제(inositol polyphosphate multikinase) 억제제를 유효성분으로 포함하는 암의 예방 또는 치료용 약제학적 조성물에 관한 것이다.The present invention relates to a pharmaceutical composition for preventing or treating cancer comprising an inositol polyphosphate multikinase inhibitor as an active ingredient.
암은 인간의 건강을 위협하는 가장 흔하고 심각한 질병이며, 항암제의 연구와 개발은 암환자 수명의 연장을 위해 중요하다. 최근 몇 년 동안 암 치료방법은 암유전체학과 분자약학의 급속한 발전과 새로운 항암제 개발로 많은 발전을 이루었지만, 여전히 새로운 치료제의 개발이 필요하다. 대장암은 맹장, 결장과 직장에 생기는 악성 종양으로 대장의 가장 안쪽 표면인 점막에서 발생한다. 대장암은 우리나라에서 위암에 이어 두 번째로 흔히 발생하는 암으로 근래에 식생활의 양상이 서구화되어 가면서 그 발생 빈도가 가파르게 증가하고 있고, 최근 10년 사이 대장암에 의한 사망률은 약 80%정도 증가하여 그 상승속도가 계속 높아지고 있는 추세이다. 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 to develop 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. In recent years, the incidence of colorectal cancer has increased sharply with the westernization of diet. Its rate of increase continues to increase.
대장암의 치료는 외과적 절제, 항암 약물치료, 방사선 치료로 나눌 수 있다. 대장암의 전 단계인 용종이나 용종에 국한된 초기의 대장·직장암의 경우에는 용종절제술등으로 치료가 가능하다. 대장암의 치료에는 1970년대 개발된 플루오로우라실(Fluorouracil, 5-FU) 이후로 이리노테칸(irinotecan), 옥살리플라틴(oxaliplatin), 카페시타빈(capecitabine), TAS-102 등의 5개 항암제와 상피세포 성장인자 수용체(epidermal growth factor receptor, EGFR)나 혈관 내피 성장인자(vascular endothelial growth factor, VEGF), 혈관 내피 성장인자 수용체(VEGF-R)를 표적으로 하는 표적 항암제로 세툭시맙(cetuximab), 파니투무맙(panitumumab), 베바시주맙(bevacizumab), 애프리버셉트(aflibercept), 레고라페닙(regorafenib) 등의 5개 표적항암제가 미국 FDA에 인정받아 사용되고 있다. 그러나 여전히 항암 효과, 안정성, 체내 흡수 효과가 우수한 대장암 치료제의 개발이 시급한 실정이다. Treatment of colorectal cancer can be divided into surgical resection, chemotherapy, and radiation therapy. Polyps, which are the previous stages of colorectal cancer, or early colon/rectal cancer limited to polyps, can be treated with polypectomy. For the treatment of colorectal cancer, since Fluorouracil (5-FU) developed in the 1970s, five anticancer drugs such as irinotecan, oxaliplatin, capecitabine, and TAS-102 and epithelial cell growth cetuximab, Panitu, as 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.
본 발명자들은 암세포 성장의 억제와 암세포 사멸을 위한 화합물을 개발하고자 예의 연구 노력하였다. 그 결과 이노시톨 다인산 멀티키나아제(Inositol Polyphosphate Multikinase, IPMK) 억제제를 유효성분으로 포함하는 조성물의 경우 암 치료에 효과가 있다는 것을 규명함으로써, 본 발명을 완성하게 되었다. The present inventors made intensive research efforts to develop compounds for inhibiting cancer cell growth and killing cancer cells. As a result, in the case of a composition comprising an Inositol Polyphosphate Multikinase (IPMK) inhibitor as an active ingredient, it was found that it is effective in cancer treatment, thereby completing the present invention.
따라서, 본 발명의 목적은 IPMK 억제제를 유효성분으로 포함하는 암의 예방 또는 치료용 약제학적 조성물을 제공하는 것이다.Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating cancer comprising an IPMK inhibitor as an active ingredient.
본 발명의 일 양태에 따르면, 본 발명은 이노시톨 다인산 멀티키나아제(inositol polyphosphate multikinase) 억제제를 유효성분으로 포함하는 암의 예방 또는 치료용 약제학적 조성물을 제공한다.According to one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating cancer comprising an inositol polyphosphate multikinase inhibitor as an active ingredient.
이노시톨 다인산(inositol polyphosphate, IP)은 일련의 이노시톨 키나아제들에 의해 이노시톨 삼인산(inositol 1,4,5-trisphosphate, IP3)이 순차적으로 인산화되어 생합성되어 합성되는 이노시톨 대사물질로서, 이노시톨 링구조에 결합하는 인산기의 숫자에 따라 IP4, IP5, IP6로 불린다. 특히 두 개의 인산기가 같이 이중으로 결합되는 경우, IP7 및 IP8의 합성이 가능하며 이러한 종류의 이노시톨 다인산은 특별히 이노시톨 파이로인산이라고 불린다. Inositol polyphosphate (IP) is an inositol metabolite synthesized by sequential phosphorylation of inositol 1,4,5-trisphosphate (IP3) by a series of inositol kinases and biosynthesis, and is bound to the inositol ring structure It is called IP4, IP5, or IP6 depending on the number of phosphate groups. In particular, when two phosphate groups are double bonded together, IP7 and IP8 can be synthesized, and this type of inositol polyphosphate is specifically called inositol pyrophosphate.
이노시톨 다인산 멀티키나아제(Inositol Polyphosphate Multikinase Inhibitor, IPMK)는 세포 내에서 이노시톨 다인산의 생합성에 필수적인 대사효소로서 세포의 성장 및 대사반응에 관여하는 단백질이다. 따라서 IPMK가 제거 또는 억제된 세포에서는 IP5, IP6, IP7과 같은 이노시톨 다인산들이 정상적으로 합성되지 않는다. 또한, IPMK는 PIP3 의존적인 Akt 신호경로의 활성화를 조절하는 기능을 수행한다. Inositol polyphosphate multikinase (IPMK) is an essential metabolic enzyme for the biosynthesis of inositol polyphosphate in cells, and is a protein involved in cell growth and metabolic reactions. Therefore, inositol polyphosphates such as IP5, IP6, and IP7 are not normally synthesized in cells in which IPMK is removed or suppressed. In addition, IPMK functions to regulate the activation of the PIP3-dependent Akt signaling pathway.
IPMK 억제제는 IPMK 자체의 활성을 억제하거나 IPMK의 활성을 높이는 상위단계의 인자의 활성을 억제하는 물질을 의미할 수 있다. 또한, IPMK 억제제는 IPMK의 활성을 억제하여, Akt 신호경로의 활성화를 조절하고, Akt 473 인산화의 감소를 통해 암세포의 성장을 억제하는 효과가 있다. 따라서, IPMK 억제제는 다양한 암종에 대해 항암 효과를 나타낼 수 있다. The IPMK inhibitor may refer to a substance that inhibits the activity of IPMK itself or inhibits the activity of an upper-level factor that increases the activity of IPMK. In addition, the IPMK inhibitor has the effect of inhibiting the activity of IPMK, regulating the activation of the Akt signaling pathway, and inhibiting the growth of cancer cells through reduction of Akt 473 phosphorylation. Therefore, IPMK inhibitors may exhibit anticancer effects against various carcinomas.
본 발명의 일 구현예에 있어서, 상기 이노시톨 다인산 멀티키나아제 억제제는 빌라조돈(vilazodone)이다. 빌라조돈(상품명 Viibryd)은 독일 머크사(Merck)에서 개발한 항우울제로 사용되는 약이며, 미국은 빌라조돈의 항우울제 용도를 2011년에 승인하였다. In one embodiment of the present invention, the inositol polyphosphate multikinase inhibitor is vilazodone. Villazodone (trade name: Viibryd) is a drug used as an antidepressant developed by Merck in Germany, and the US approved the use of vilazodone as an antidepressant in 2011.
상기 빌라조돈의 IUPAC 명은 5-(4-[4-(5-시아노-1H-인돌-3-yl)부틸]피페라진-1-yl)벤조푸란-2-카르복스아미드(5-(4-(4-(5-Cyano-1H-indol-3-yl)butyl)piperazin-1-yl)benzofuran-2-carboxamide)이다. The IUPAC name of vilazodone is 5-(4-[4-(5-cyano-1H-indole-3-yl)butyl]piperazin-1-yl)benzofuran-2-carboxamide (5-(4 -(4-(5-Cyano-1H-indol-3-yl)butyl)piperazin-1-yl)benzofuran-2-carboxamide).
본 발명의 일 구체예에 있어서, 상기 이노시톨 다인산 멀티키나아제 억제제는 빌라조돈, 이의 약제학적으로 허용되는 염 또는 이의 광학이성질체이다.In one embodiment of the present invention, the inositol polyphosphate multikinase inhibitor is vilazodone, a pharmaceutically acceptable salt thereof, or an optical isomer thereof.
본 발명의 일 구현예에 있어서, 상기 암은 고형암이다.In one embodiment of the present invention, the cancer is a solid cancer.
본 명세서 상의 용어 "고형암"이란 혈액암과는 구별되는 특징을 지니고, 방광, 유방, 장, 신장, 폐, 간, 뇌, 식도, 쓸개, 난소, 췌장, 위, 자궁경부, 갑상선, 전립선 및 피부 등의 여러 고형 장기(solid organ)에서 비정상적으로 세포가 성장하여 발생한 덩어리로 이루어진 암이다. 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
본 발명의 일 구체예에 있어서, 상기 고형암은 뇌종양, 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 상의세포종, 뇌간종양, 두경부 종양, 후두암, 구인두암, 비강/부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 구강암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 남성유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 직장암, 항문암, 방광암, 신장암, 남성 생식기종양, 음경암, 전립선암, 여성생식기종양, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성외부생 식기암, 여성요도암 및 피부암으로 이루어지는 군으로부터 선택되는 것이나 이에 한정되는 것은 아니다.In one embodiment of the present invention, the solid cancer is 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 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, ectopic It is selected from the group consisting of gastric cancer, female urethral cancer, and skin cancer, but is not limited thereto.
본 발명의 일 구현예에 있어서, 상기 암은 혈액암이다. In one embodiment of the present invention, the cancer is a hematologic cancer.
본 명세서 상의 용어 "혈액암"이란 혈액을 구성하는 성분에 생긴 암을 지칭하는 것으로, 혈액, 조혈기관, 림프절, 림프기관 등에 발생한 악성 종양을 의미한다. 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.
본 발명의 일 구체예에 있어서, 상기 혈액암은 급성골수구성백혈병, 급성림프구성 백혈병, 만성골수성백혈병, 만성림프구성백혈병, 급성단구성백혈병, 다발성 골수종, 호지킨림프종 및 비호지킨 림프종으로 이루어진 군으로부터 선택되는 것이나 이에 한정되는 것은 아니다. In one embodiment of the present invention, the hematologic cancer is acute myelocytic 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.
본 발명의 약제학적 조성물은 유효성분인 상기 조성물 외에 약제학적으로 허용되는 담체를 포함할 수 있다.The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier in addition to the composition as an active ingredient.
본 발명의 약제학적 조성물에 포함되는 약제학적으로 허용되는 담체는 제제시에 통상적으로 이용되는 것으로서, 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아 고무, 인산 칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세결정성 셀룰로스, 폴리비닐피롤리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함하나, 이에 한정되는 것은 아니다. 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; it is not going to be
본 발명의 약제학적 조성물은 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다. 적합한 약제학적으로 허용되는 담체 및 제제는 Remington's Pharmaceutical Sciences (19th ed., 1995)에 상세히 기재되어 있다.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).
본 발명의 약제학적 조성물은 경구 또는 비경구로 투여할 수 있고, 예컨대 척추강 내 투여, 정맥내 투여, 피하 투여, 피내 투여, 근육내 투여, 복강내 투여, 흉골 내 투여, 종양 내 투여, 비내 투여, 뇌내 투여, 두개골 내 투여, 폐내 투여 및 직장내 투여 등으로 투여할 수 있으나 이에 한정되는 것은 아니다.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.
본 발명의 약제학적 조성물의 적합한 투여량은 제제화 방법, 투여방식, 환자의 연령, 체중, 성, 병적 상태, 음식, 투여 시간, 투여 경로, 배설 속도 및 반응 감응성과 같은 요인들에 의해 다양하며, 보통으로 숙련된 의사는 소망하는 치료 또는 예방에 효과적인 투여량(약제학적 유효량)을 용이하게 결정 및 처방할 수 있다. 본 발명의 바람직한 구현예에 따르면, 본 발명의 약제학적 조성물의 1일 투여량은 0.0001-100 ㎎/㎏이다. A suitable dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, 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.
본 명세서에서 용어 "약제학적 유효량"은 상술한 질환을 예방 또는 치료하는 데 충분한 양을 의미한다.As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to prevent or treat the above-mentioned diseases.
본 명세서에서 용어 “예방”은 질환 또는 질환 상태의 방지 또는 보호적인 치료를 의미한다. 본 명세서에서 용어 “치료”는 질환 상태의 감소, 억제, 진정 또는 근절을 의미한다.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.
본 발명의 약제학적 조성물은 당해 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 용이하게 실시할 수 있는 방법에 따라, 약제학적으로 허용되는 담체 및/또는 부형제를 이용하여 제제화 함으로써 단위 용량 형태로 제조되거나 또는 다용량 용기 내에 내입시켜 제조될 수 있다. 이때 제형은 내복약, 주사제 등 다양하게 제조될 수 있고, 오일 또는 수성 매질중의 용액, 현탁액 또는 유화액 형태이거나 엑스제, 산제, 좌제, 분말제, 과립제, 정제 또는 캅셀제 형태일 수도 있으며, 분산제 또는 안정화제를 추가적으로 포함할 수 있다.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 can 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.
본 발명의 실시예에 따르면, 본 발명의 빌라조돈을 포함하는 약제학적 조성물은 IPMK의 활성을 억제하는 효과 및 세포의 자가포식기작에 관여하는 단백질의 발현을 증가시키는 효과가 있다. According to an embodiment of the present invention, the pharmaceutical composition comprising vilazodone of the present invention has the effect of inhibiting the activity of IPMK and increasing the expression of proteins involved in the autophagy mechanism of cells.
또한, 본 발명의 실시예에 따르면, 본 발명의 상기 약제학적 조성물은 암세포의 성장을 억제하는 효과 및 생체내에서 종양의 성장을 억제하는 효과가 있다. 따라서, 본 발명의 상기 약제학적 조성물은 암, 보다 구체적으로는 대장암에 대해 항암 효과가 있다. 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 has an anticancer effect on cancer, more specifically colon cancer.
본 발명의 다른 일 양태에 따르면, 본 발명은 상술한 본 발명의 이노시톨 다인산 멀티키나아제 억제제를 유효성분으로 포함하는 약제학적 조성물을 대상체(subject)에 투여하는 단계를 포함하는 암의 예방 또는 치료방법을 제공한다. According to another aspect of the present invention, the present invention provides a method for preventing or treating cancer comprising administering to a subject a pharmaceutical composition comprising the above-described inositol polyphosphate multikinase inhibitor of the present invention as an active ingredient provides
본 명세서에서 사용된 용어, "투여" 또는 "투여하다"는 본 발명의 조성물의 치료적, 또는 예방적 유효량을 상기 대상 질환을 겪거나, 겪을 가능성이 있는 대상체(개체)에 직접적으로 투여함으로써 대상체의 체내에서 동일한 양이 형성되도록 하는 것을 말한다. 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
상기 조성물의 "치료적 유효량"은 조성물을 투여하고자 하는 대상체에게 치료적 또는 예방적 효과를 제공하기에 충분한 조성물의 함량을 의미하며, 이에 "예방적 유효량"을 포함하는 의미이다.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 to be administered, and includes a "prophylactically effective amount".
또한, 본 명세서에서 사용된 용어, "대상체(개체)"는 인간, 마우스, 랫트, 기니아 피그, 개, 고양이, 말, 소, 돼지, 원숭이, 침팬지, 비비 및 붉은털 원숭이 등을 포함하는 포유류이다. 가장 구체적으로는, 본 발명의 대상체는 인간이다.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.
본 발명의 상기 암의 예방, 또는 치료방법은, 본 발명의 일 양태인 약제학적 조성물을 투여하는 단계를 포함하는 방법이므로, 중복되는 내용에 대해서는 본 명세서의 과도한 중복성을 피하기 위하여 그 기재를 생략한다.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. .
본 발명의 특징 및 이점을 요약하면 다음과 같다:The features and advantages of the present invention are summarized as follows:
본 발명은 이노시톨 다인산 멀티키나아제(inositol polyphosphate multikinase) 억제제를 유효성분으로 포함하는 암의 예방 또는 치료용 약제학적 조성물을 제공한다.The present invention provides a pharmaceutical composition for preventing or treating cancer comprising an inositol polyphosphate multikinase inhibitor as an active ingredient.
본 발명의 조성물은 암 세포의 성장을 억제하고 암 세포를 사멸시키는 효능을 나타내어 항암제로 사용이 가능하다. 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.
도 1a는 IP kinase assay를 통해 빌라조돈의 이노시톨 키나아제 활성 억제효과를 분석한 결과를 나타낸다. Figure 1a shows the results of analyzing the inhibitory effect of inositol kinase activity of vilazodone through IP kinase assay.
도 1b는 HPLC를 통해 NIH3T3-L1 섬유아세포에 대하여 DMSO, 빌라조돈, 퀘르세틴을 처리한 후 IPMK의 대사산물을 분석한 결과를 나타낸다. Figure 1b shows the results of analyzing the metabolites of IPMK after treating NIH3T3-L1 fibroblasts with DMSO, vilazodone, and quercetin through HPLC.
도 1c는 웨스턴 블롯을 통해 HCT116 세포에 대하여 DMSO 약물과 빌라조돈을 처리한 후 Akt의 인산화 수준을 분석한 결과를 나타낸다. Figure 1c shows the result of analyzing the phosphorylation level of Akt after treatment with DMSO drug and vilazodone for HCT116 cells through Western blot.
도 2a는 대장암 세포주 HCT116에 각각 DMSO와 빌라조돈을 처리한 후 세포의 생존율을 분석한 결과를 나타낸다.Figure 2a shows the results of analyzing the cell viability after treating the colon cancer cell line HCT116 with DMSO and vilazodone, respectively.
도 2b는 대장암 세포주 HT29에 각각 DMSO와 빌라조돈을 처리한 후 세포의 생존율을 분석한 결과를 나타낸다.Figure 2b shows the results of analyzing the cell viability after treating the colon cancer cell line HT29 with DMSO and vilazodone, respectively.
도 2c는 대장암 세포주 SW480에 각각 DMSO와 빌라조돈을 처리한 후 세포의 생존율을 분석한 결과를 나타낸다.Figure 2c shows the results of analyzing the cell viability after treating the colon cancer cell line SW480 with DMSO and vilazodone, respectively.
도 3은 웨스턴 블롯을 통해 대표 대장암 세포주 3 종 (HCT116, HT29, SW480)에 대하여 DMSO와 빌라조돈을 처리한 후 세포의 자가포식과 관련된 단백질을 분석한 결과를 나타낸다.3 shows the results of analysis of proteins related to autophagy of cells after treatment with DMSO and vilazodone for three representative colorectal cancer cell lines (HCT116, HT29, SW480) through Western blot.
도 4는 대장암 세포 HT29에 대하여 DMSO와 빌라조돈을 처리한 후 암세포 콜로니 증식 정도를 측정한 결과를 나타낸다.4 shows the results of measuring the degree of cancer cell colony proliferation after treatment with DMSO and vilazodone for colorectal cancer cells HT29.
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 보다 구체적으로 설명하기 위한 것으로, 본 발명의 요지에 따라 본 발명의 범위가 이들 실시예에 의해 제한되지 않는다는 것은 당업계에서 통상의 지식을 가진 자에 있어서 자명할 것이다.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. .
실시예Example
본 명세서 전체에 걸쳐, 특정 물질의 농도를 나타내기 위하여 사용되는 "%"는 별도의 언급이 없는 경우, 고체/고체는 (중량/중량) %, 고체/액체는 (중량/부피) %, 그리고 액체/액체는 (부피/부피) %이다.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) %.
실시예 1: 세포의 배양 방법Example 1: Cell culture method
HCT116 세포(인간 결장암 세포, p53 야생형), HT29 세포(인간 결장암 세포, p53 돌연변이체), SW480 세포(인간 결장암 세포, p53 돌연변이체)를 ATCC(American Type Culture Collection, Virginia, USA)로부터 구입하여 사용하였다.HCT116 cells (human colon cancer cells, p53 wild-type), HT29 cells (human colon cancer cells, p53 mutant), and SW480 cells (human colon cancer cells, p53 mutant) were purchased from ATCC (American Type Culture Collection, Virginia, USA) and used. did
HCT116 세포는 2 mM 글루타민, 1% 페니실린-스트렙토마이신 및 10% 소태아 혈청(FBS)이 보충된 McCoy's 5a 배지(Sigma Aldrich, Missouri, USA)에서 37 ℃, 5% CO2의 조건으로 배양하였다. HT29, SW480 세포는 2 mM 글루타민, 1% 페니실린-스트렙토마이신 및 10% 소태아 혈청(FBS)이 보충된 RPMI 배지(Sigma Aldrich, Missouri, USA)에서 37 ℃, 5% CO2의 조건으로 배양하였다. 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. 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. .
실시예 2: 빌라조돈의 IPMK 억제 효과 확인Example 2: Confirmation of IPMK inhibitory effect of vilazodone
실시예 2-1. IP kinase assay를 수행하여 확인한 빌라조돈의 IPMK 억제 효과Example 2-1. IPMK inhibitory effect of vilazodone confirmed by performing IP kinase assay
빌라조돈의 IPMK 억제효과는 ADP-Glo Kinase kit(Promega, Wisconsin, USA)를 사용하여 측정하였다. The IPMK inhibitory effect of vilazodone was measured using the ADP-Glo Kinase kit (Promega, Wisconsin, USA).
96 웰 플레이트에 kinase assay buffer(50 mM HEPES [pH 7.4], 10 mM MgCl2, 50 mM KCl), 50 ng의 재조합 IPMK, 10 μM ATP, 및 100 μM의 빌라조돈 또는 퀘르세틴(quercetin)(Sigma Aldrich, Missouri, USA)을 첨가하여 실험을 수행하였다. 반응을 ADP-Glo 시약으로 실온에서 40 분동안 퀜치하였다. 그 후, 키나아제 검출 기질을 첨가하고, 반응물을 추가로 30 분동안 배양하였다. 플레이트 판독으로 발광을 검출하였다. In 96-well plates, kinase assay buffer (50 mM HEPES [pH 7.4], 10 mM MgCl2, 50 mM KCl), 50 ng recombinant IPMK, 10 μM ATP, and 100 μM vilazodone or quercetin (Sigma Aldrich, Missouri, USA) was added to the experiment. The reaction was quenched with ADP-Glo reagent at room temperature for 40 min. After that, kinase detection substrate was added and the reaction was incubated for an additional 30 minutes. Luminescence was detected by plate reading.
상기 실험을 통해, DMSO 대조군과 비교하여 빌라조돈이 IPMK의 효소 활성을 40 %까지 억제한다는 사실을 확인하였다(도 1a).Through the above experiment, it was confirmed that vilazodone inhibited the enzymatic activity of IPMK by 40% compared to the DMSO control (FIG. 1a).
실시예 2-2. HPLC를 수행하여 확인한 빌라조돈의 IPMK 억제 효과Example 2-2. IPMK inhibitory effect of vilazodone confirmed by HPLC
NIH3T3-L1 섬유아세포(fibroblasts)(2Х105 세포)를 10 % 송아지 혈청으로 보충된 DMEM 60 mm 배지에 접종하였다. 세포를 60 μCi [3H]-마이오-이노시톨로 3일 동안 표지하였다. 빌라조돈과 퀘르세틴은 0.1 %의 농도가 되도록 DMSO에 용해하였으며, DMSO에 용해된 빌라조돈 또는 퀘르세틴 10 μM을 실험을 위해 처리하였다. 가용성 이노시톨 대사 산물은 과염소산 완충액으로 추출하고, 나머지 불용성 이노시톨 대사 산물은 0.1 M NaOH 및 0.1% Triton X-100를 사용하여 추출하였다. 3H-표지된 IP를 고성능 액체 크로마토그래피(HPLC)로 분석하고, 각 분획을 액체섬광계수기(liquid scintillation counter)를 통해 측정하였다. NIH3T3-L1 fibroblasts (2Х10 5 cells) were inoculated in DMEM 60 mm medium supplemented with 10% calf serum. Cells were labeled with 60 μCi [ 3 H]-myo-inositol for 3 days. Villazodone and quercetin were dissolved in DMSO to a concentration of 0.1%, and 10 μM of vilazodone or quercetin dissolved in DMSO was treated for the experiment. Soluble inositol metabolites were extracted with perchloric acid buffer, and the remaining insoluble inositol metabolites were extracted using 0.1 M NaOH and 0.1% Triton X-100. 3H-labeled IP was analyzed by high performance liquid chromatography (HPLC), and each fraction was measured using a liquid scintillation counter.
상기 실험을 통해, 빌라조돈이 IPMK에 의해 합성되는 인산화IP 대사산물인 IP5, IP6, IP7의 수준을 억제한다는 사실을 확인하였다(도 1b). Through the above experiment, it was confirmed that vilazodone inhibits the levels of IP5, IP6, and IP7, which are phosphorylated IP metabolites synthesized by IPMK (FIG. 1b).
이러한 결과는 빌라조돈이 IPMK의 활성을 억제하는 효과를 갖고 있음을 나타낸다.These results indicate that vilazodone has the effect of inhibiting the activity of IPMK.
실시예 2-3. 면역블롯팅을 수행하여 확인한 빌라조돈의 IPMK 억제 효과Example 2-3. IPMK inhibitory effect of vilazodone confirmed by immunoblotting
HCT116 세포를 6 웰 플레이트에 4Х104 세포/웰의 밀도로 접종하고 하루 뒤에 10 μM의 빌라조돈을 처리하였다. 빌라조돈 처리 4시간 후에 세포를 NP-40 버퍼(50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM EDTA [pH 8.0], 50 mM NaF, 10 mM Na-파이로인산염, 및 단백질 억제제 칵테일 [Roche, Switzerland])로 용해하였다. 세포용해물을 4 내지 12 %의 Bis-Tris SDS-PAGE 겔에 로딩하고, 니트로셀룰로오스막에 트렌스퍼하였다. 포스포-S473 Akt, 토탈 Akt (Cell Signaling Technology, Massachusetts, USA), 및 α-튜불린 (Sigma Aldrich, Missouri, USA) 항체는 1:1,000으로 희석하여 사용하였다. 2차 항체는 1:2,000으로 희석하여 사용하였다. 단백질은 화학발광 면역블롯 검출 시스템(Bio-Rad, California USA)을 사용하여 검출하였다.HCT116 cells were seeded in a 6-well plate at a density of 4Х10 4 cells/well and treated with 10 μM of vilazodone one day later. After 4 h of vilazodone treatment, cells were washed with NP-40 buffer (50 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% NP-40, 1 mM EDTA [pH 8.0], 50 mM NaF, 10 mM Na-Pi). lysate, and a cocktail of protein inhibitors [Roche, Switzerland]). The cell lysate was loaded on a 4 to 12% Bis-Tris SDS-PAGE gel, and transferred to a nitrocellulose membrane. Phospho-S473 Akt, Total Akt (Cell Signaling Technology, Massachusetts, USA), and α-tubulin (Sigma Aldrich, Missouri, USA) antibodies were diluted 1:1,000 and used. The secondary antibody was diluted 1:2,000 and used. Proteins were detected using a chemiluminescent immunoblot detection system (Bio-Rad, California USA).
상기 실험을 통해, 빌라조돈이 HCT116세포에서 Akt의 인산화를 억제한다는 사실을 확인하였으며 (도 1c), 이는 빌라조돈이 Akt 활성과 관련된 IPMK의 작용을 억제하는 것을 나타낸다. Through the above experiment, it was confirmed that vilazodone inhibited the phosphorylation of Akt in HCT116 cells (FIG. 1c), which indicates that vilazodone inhibits the action of IPMK related to Akt activity.
실시예 3: 빌라조돈이 처리된 세포의 생존력 분석(viability assay)Example 3: Viability assay of vilazodone-treated cells
HCT116, HT29, SW480 세포를 96 웰 플레이트에 104 세포/웰의 밀도로 접종하고, 24시간 동안 37 ℃에서 배양한 후, 다양한 농도(0, 1.25, 2.5, 5, 10, 20 μM)의 빌라조돈(Selleckchem, Texas, USA)로 처리하였다. 빌라조돈 처리 후 플레이트를 5% CO2, 37 ℃에서 24, 48, 72시간 동안 배양하였다. 그 후, 세포를 분석 시약 100 μL을 각 웰에 첨가한 후 VICTOR X Multilabel Reader (PerkinElmer, Massachusetts, USA)를 사용하여 발광(luminescence)을 측정하여 도 2에 나타내었다.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, 1.25, 2.5, 5, 10, 20 μM) of Villa It was treated with crude pigs (Selleckchem, Texas, USA). After treatment with vilazodone Plates were incubated at 5% CO 2 , 37° C. for 24, 48, 72 hours. Thereafter, 100 μL of an assay reagent was added to each well of the cells, and luminescence was measured using a VICTOR X Multilabel Reader (PerkinElmer, Massachusetts, USA), and is shown in FIG. 2 .
상기 실험을 통해, 대표 대장암 세포주 3 종 (HCT116, HT29, SW480)에서 빌라조돈의 용량이 증가할수록 세포의 생존율이 감소함을 확인하였다(도 2).Through the above experiment, it was confirmed that the cell viability decreased as the dose of vilazodone increased in three representative colorectal cancer cell lines (HCT116, HT29, SW480) ( FIG. 2 ).
상기 결과는 빌라조돈이 다양한 대장암 세포를 사멸시키는데 매우 효과적인 것을 나타낸다. The above results indicate that vilazodone is very effective in killing various colorectal cancer cells.
실시예 4: 빌라조돈이 처리된 세포의 자가포식 분석(autophagy assay)Example 4: Autophagy assay of vilazodone-treated cells
HCT116, HT29, SW480 세포주에서의 빌라조돈의 세포 자가포식 기능의 연관성을 확인하기 위해, 빌라조돈 첨가 시 세포의 신호전달 효과를 살펴보았다.In order to confirm the correlation between the cellular autophagy function of vilazodone in HCT116, HT29, and SW480 cell lines, the signaling effect of cells upon addition of vilazodone was examined.
세포 자가포식에 연관된 세포 신호전달 변화 분석을 위한 다양한 농도(0, 5, 10 μM)로 빌라조돈이 처리된 HCT116, HT29, SW480 세포를 웨스턴 블롯으로 분석하였다. 웨스턴 블롯을 위해 수득된 상등액을 10% SDS-PAGE 젤에 로딩하여 분리하고, 분리된 단백질을 PVDF 막에 블로팅하였다. 그 다음 블롯에 항-LC3, 항-p-ULK1 항체(Cell Signaling Technology)를 첨가하고, 4 ℃에서, 12시간 동안 인큐베이션하였다. 그 후 블롯을 TBST (a mixture of tris-buffered saline (TBS) and Tween 20)로 세척하고 호스래디쉬 퍼옥시다제-결합(horseradish peroxidase-conjugated) 2차 항체(Cell signaling)와 37 ℃에서, 1시간 동안 인큐베이션하고 세척한 후, 강화된 화학발광(ECL; Amersham)을 검출하였다. Villazodone-treated at various concentrations (0, 5, 10 μM) for analysis of cellular signaling changes associated with cellular autophagy. HCT116, HT29, SW480 cells were analyzed by Western blot. The supernatant obtained for Western blotting was separated by loading on a 10% SDS-PAGE gel, and the separated protein was blotted on a PVDF membrane. Then, anti-LC3, anti-p-ULK1 antibody (Cell Signaling Technology) was 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.
상기 실험을 통해, 빌라조돈은 농도 의존적으로 ULK1의 인산화 수준을 현저히 감소시키며 LC3I의 LC3II로의 유도를 뚜렷이 증가시킴을 확인하였다 (도 3). 이러한 결과는 빌라조돈이 자가포식 기작에 관여하는 단백질의 발현을 크게 증가시키는 효과를 갖고 있음을 나타낸다. Through the above experiment, it was confirmed that vilazodone significantly decreased the phosphorylation level of ULK1 and significantly increased the induction of LC3I to LC3II in a concentration-dependent manner ( FIG. 3 ). These results indicate that vilazodone has the effect of significantly increasing the expression of proteins involved in the autophagy mechanism.
실시예 5: 빌라조돈이 처리된 세포의 콜로니 증식 분석(cell colony forming assay)Example 5: Assay for colony growth of vilazodone-treated cells (cell colony forming assay)
HT29 세포주에서 빌라조돈과 암세포 콜로니 증식율의 연관성을 확인하기 위해, HT29 세포를 배양하는 웰에 빌라조돈 첨가 시 암세포 콜로니 증식율을 살펴보았다.In order to confirm the correlation between vilazodone and cancer cell colony growth rate in the HT29 cell line, the cancer cell colony growth rate was examined when vilazodone was added to the wells culturing HT29 cells.
HT29 세포를 12 웰 플레이트에 105 세포/웰의 밀도로 접종하고 24시간 동안 37℃에서 배양한 후 세포를 0, 5, 10, 20 μM 농도의 빌라조돈으로 처리하였다. 빌라조돈 처리 후 플레이트를 5% CO2, 37 ℃에서 48시간 동안 배양하였다. 그 후, 크리스탈 바이올렛 500 μL을 각 웰에 첨가한 후 10분동안 상온에서 세포를 염색하여 이로부터 세포의 증식력을 분석하였다.HT29 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, 10, 20 μM vilazodone. After treatment with vilazodone 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.
실험을 통해, 빌라조돈을 처리한 웰에서의 세포 증식율이 빌라조돈을 처리하지 않은 웰에서의 증식율보다 현저히 낮다는 것을 확인하였다 (도 4). Through the experiment, it was confirmed that the cell proliferation rate in wells treated with vilazodone was significantly lower than that in wells not treated with vilazodone (FIG. 4).
이러한 결과는 빌라조돈이 매우 뛰어난 항암 효과를 갖고 있음을 나타낸다.These results indicate that vilazodone has a very good anticancer effect.

Claims (7)

  1. 이노시톨 다인산 멀티키나아제(inositol polyphosphate multikinase) 억제제를 유효성분으로 포함하는 암의 예방 또는 치료용 약제학적 조성물.A pharmaceutical composition for preventing or treating cancer comprising an inositol polyphosphate multikinase inhibitor as an active ingredient.
  2. 제1항에 있어서, 상기 이노시톨 다인산 멀티키나아제 억제제는 빌라조돈(vilazodone)인 것인, 암의 예방 또는 치료용 약제학적 조성물. The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the inositol polyphosphate multikinase inhibitor is vilazodone.
  3. 제1항에 있어서, 상기 암은 고형암인 것인, 암의 예방 또는 치료용 약제학적 조성물.The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is a solid cancer.
  4. 제3항에 있어서, 상기 고형암은 뇌종양, 양성성상세포종, 악성성상세포종, 뇌하수체 선종, 뇌수막종, 뇌림프종, 핍지교종, 상의세포종, 뇌간종양, 두경부 종양, 후두암, 구인두암, 비강/부비동암, 비인두암, 침샘암, 하인두암, 갑상선암, 구강암, 흉부종양, 소세포성 폐암, 비소세포성 폐암, 흉선암, 종격동 종양, 식도암, 유방암, 남성유방암, 복부종양, 위암, 간암, 담낭암, 담도암, 췌장암, 소장암, 대장암, 직장암, 항문암, 방광암, 신장암, 남성 생식기종양, 음경암, 전립선암, 여성생식기종양, 자궁경부암, 자궁내막암, 난소암, 자궁육종, 질암, 여성외부생 식기암, 여성요도암 및 피부암으로 이루어지는 군으로부터 선택되는 것인, 암의 예방 또는 치료용 약제학적 조성물.According to claim 3, wherein the solid cancer is brain tumor, benign astrocytoma, malignant astrocytoma, pituitary adenoma, meningioma, cerebral 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, mediastinum tumor, esophageal cancer, breast cancer, male breast cancer, abdominal tumor, stomach cancer, liver cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, small intestine cancer 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, external genital cancer, A pharmaceutical composition for preventing or treating cancer, which will be selected from the group consisting of female urethral cancer and skin cancer.
  5. 제1항에 있어서, 상기 암은 혈액암인 것인, 암의 예방 또는 치료용 약제학적 조성물.The pharmaceutical composition for preventing or treating cancer according to claim 1, wherein the cancer is a blood cancer.
  6. 제5항에 있어서, 상기 혈액암은 급성골수구성백혈병, 급성림프구성 백혈병, 만성골수성백혈병, 만성림프구성백혈병, 급성단구성백혈병, 다발성 골수종, 호지킨림프종 및 비호지킨 림프종으로 이루어진 군으로부터 선택되는 것인, 암의 예방 또는 치료용 약제학적 조성물. 6. The method of claim 5, wherein the hematologic cancer is selected from the group consisting of acute myelocytic 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.
  7. 이노시톨 다인산 멀티키나아제 억제제를 유효성분으로 포함하는 약제학적 조성물을 대상체(subject)에 투여하는 단계를 포함하는 암의 예방 또는 치료방법.A method for preventing or treating cancer, comprising administering to a subject a pharmaceutical composition comprising an inositol polyphosphate multikinase inhibitor as an active ingredient.
PCT/KR2021/003295 2020-05-26 2021-03-17 Pharmaceutical composition for cancer prevention or treatment, comprising inositol polyphosphate multikinase inhibitor as active ingredient WO2021241862A1 (en)

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