KR102656587B1 - Pharmaceutical composition for the death of cancer origin cell - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for killing cancer origin cells, comprising perphenazine, a biguanide compound, and a glucose uptake inhibitor as active ingredients.
The pharmaceutical composition of the present invention can not only effectively improve or treat cancer by specifically killing cancer origin cells, but can also prevent the onset of cancer itself or prevent cancer recurrence.

Description

Pharmaceutical composition for killing cancer origin cells {Pharmaceutical composition for the death of cancer origin cell}

The present invention relates to a pharmaceutical composition that can not only fundamentally treat cancer by killing cancer origin cells, but also prevent the onset or recurrence of cancer.

Cancer is also called a tumor, as it is a cell mass composed of undifferentiated cells that proliferate indefinitely, ignoring the necessary conditions within the tissue, unlike normal cells that can proliferate and suppress in a regular and controlled manner according to the individual's needs. Cancer cells that proliferate indefinitely infiltrate surrounding tissues and, in more serious cases, metastasize to other organs of the body, causing severe pain and ultimately death, making it an incurable disease.

According to data from the American Cancer Society, in 2007 alone, more than 12 million patients were newly diagnosed with cancer worldwide, and the number of deaths was 7.6 million, with approximately 20,000 people dying from cancer every day. In Korea, according to the 2006 National Statistical Office report, death due to cancer ranked first as the cause of death. Therefore, the development of tumor therapeutics with excellent therapeutic effects is urgently needed to reduce mental and physical pain caused by developing and fighting cancer and improve quality of life.

However, despite much effort, the exact mechanism by which normal cells are transformed into cancer cells has not yet been clearly identified. However, external factors such as environmental factors, chemicals, radiation, viruses, and internal factors such as genetic factors and immunological factors have not yet been identified. Cancer occurs as a result of a complex intertwining of factors. Genes involved in the development of cancer include oncogenes and tumor suppressor genes, and cancer occurs when the balance between them is broken by the internal or external factors described above.

Cancer is broadly classified into blood cancer and solid cancer, and occurs in almost all parts of the body, including lung cancer, stomach cancer, breast cancer, oral cancer, liver cancer, uterine cancer, esophageal cancer, and skin cancer. Recently, a few targets such as Gleevec or Herceptin have been used as treatment methods for these cancers. Treatments are being used to treat certain cancers, but to date, surgery, radiation therapy, and anticancer treatment using chemotherapy agents that inhibit cell proliferation are the main methods. However, because they are not targeted treatments, the biggest problems with existing chemotherapy drugs are side effects due to cytotoxicity and drug resistance, which are major factors that ultimately cause treatment to fail despite an initial successful response to anticancer drugs. Therefore, in order to overcome the limitations of these chemotherapy drugs, the development of targeted treatments with a clear anticancer action mechanism is continuously needed.

Meanwhile, glioma is a tumor that accounts for 60% of primary brain tumors. It is a malignant tumor that occurs frequently and is difficult to treat, and there is no specific treatment other than radiation therapy to date. Among them, glioblastoma (GBM), which is classified as the most malignant, has a very high resistance to radiation and anticancer drug treatment compared to other cancers, and once diagnosed, the survival period is only one year, so each patient's Proper diagnosis and understanding of the origin and course are important.

In addition, in the case of brain tumors, not only is it difficult for drugs for treatment to be delivered to the target brain area due to the presence of the brain blood barrier, but also the development of treatments is difficult due to the relative lack of understanding of brain neurobiology. The reality is that it is not active. Moreover, compared to other brain tumors, glioblastoma shows an aggressive variant, which can lead to fatal results within a few weeks if not treated quickly.

Therefore, in the treatment of glioblastoma, in addition to surgical treatment, radiation therapy and chemical drug treatment are performed together. However, there is no perfect treatment due to causes such as the occurrence of resistance mutations and recurrence due to tumor stem cells.

It has recently been revealed that the cells of origin of the glioblastoma correspond to cells in the subventricular zone and cells that migrated from the subventricular zone (Korean Patent Publication No. 10-2019-0095074). Therefore, for effective treatment of glioblastoma, there is a need for early diagnosis and understanding of the origin of glioblastoma and development of new treatments based on it.

One object of the present invention is to provide a pharmaceutical composition that can not only fundamentally treat cancer by killing cancer origin cells, but also prevent the onset or recurrence of cancer.

However, the technical problem to be achieved by the present invention is not limited to the problems mentioned above, and other problems not mentioned can be clearly understood by those skilled in the art from the description below.

According to one embodiment of the present invention, it relates to a pharmaceutical composition for killing cancer origin cells.

According to another embodiment of the present invention, it relates to a pharmaceutical composition for preventing or treating cancer.

The pharmaceutical composition provided by the present invention,

(1) perphenazine or a pharmaceutically acceptable salt thereof;

(2) Biguanide-based compounds or pharmaceutically acceptable salts thereof; and

(3) A glucose uptake inhibitor or a pharmaceutically acceptable salt thereof may be included as an active ingredient.

In the present invention, “perphenazine” is a mental stabilizer and belongs to the dopamine 3 receptor antagonist (dopamine 3 receptor anatagonist). The perphenazine is represented by the structure of Formula 1 below, and the compound name is 2-chloro-10-{3-[1-(2-hydroxyethyl)-4-piperazinyl]propyl}phenothiazine (2- Corresponds to chloro-10-{3-[1-(2-hydroxyethyl)-4-piperazinyl]propyl}phenothiazine). The perphenazine is available from Schering Co. under the trade name Trilafon®.

[Formula 1]

In the present invention, the biguanide-based compound is a biguanide-based diabetes treatment agent, and specific examples in the present invention include metformin, phenformin, buformin, or N-(N-( 4-(trifluoromethoxy)phenyl)carbamimidoyl)pyrrolidine-1-carboximidamide (N-(N-(4-(trifluoromethoxy)phenyl)carbamimidoyl)pyrrolidine-1-carboximidamide, "IM156" ), etc., but any biguanide-based compound that induces a nutritional deficiency-like state by interfering with intracellular energy production may be included without limitation.

In the present invention, the glucose uptake inhibitor is 2-deoxy-D-glucose, 3-bromopyruvate, 3-bromo-2- It may be 3-bromo-2-oxopropionate-1-propyl ester, 5-thioglucose, or dichloroacetic acid, but it may affect glucose metabolism in the body. Any inhibitory ingredient may be included without limitation.

In the present invention, pharmaceutically acceptable salt forms of each of the perphenazine, biguanide series compounds, and glucose absorption inhibitors may be included. Pharmaceutically acceptable salts must have low toxicity to the human body and not adversely affect the biological activity and physicochemical properties of the parent compound. Pharmaceutically acceptable salts include, but are not limited to, acid addition salts of a pharmaceutically acceptable free acid and the perphenazine, the biguanide series compound, or the glucose absorption inhibitor.

Preferred salt forms of the compounds according to the present invention include salts with inorganic acids or organic acids. At this time, the inorganic acid may be hydrochloric acid, sulfuric acid, nitric acid, phosphoric acid, perchloric acid, bromic acid, etc. In addition, organic acids include acetic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, fumaric acid, maleic acid, malonic acid, phthalic acid, succinic acid, lactic acid, citric acid, citric acid, gluconic acid, tartaric acid, salicylic acid, malic acid, Oxalic acid, benzoic acid, embonic acid, aspartic acid, glutamic acid, etc. can be used. Organic bases that can be used to prepare organic base addition salts include tris(hydroxymethyl)methylamine and dicyclohexylamine. Amino acids that can be used to produce amino acid addition salts are natural amino acids such as alanine and glycine. It will be apparent to those skilled in the art that other acids or bases may be used in addition to the inorganic acids, organic acids, organic bases, and amino acids exemplified above.

In the present invention, the salt form can be prepared by conventional methods. For example, the above-described perphenazine, biguanide compounds, or glucose absorption inhibitor compounds are dissolved in a water-miscible solvent such as methanol, ethanol, acetone, or 1,4-dioxane, and then dissolved in a free acid or base. It can be manufactured by crystallizing it after adding it.

In the pharmaceutical composition of the present invention, any two compounds of perphenazine, biguanide series compounds and glucose absorption inhibitors are used in an amount of 1:0.001 to 1:1000, preferably 1:0.01 to 1:100, more preferably It can be used at a molar concentration ratio of 1:0.1 to 1:10, but is not limited thereto.

The pharmaceutical composition of the present invention can not only effectively improve or treat cancer by specifically killing the origin cells of cancer cells, but can also prevent the onset of cancer itself or prevent recurrence of cancer.

In the present invention, the term "cancer" typically refers to or refers to a physiological condition characterized by uncontrolled cell growth, and depending on the site of occurrence, brain cancer, ovarian cancer, colon cancer, pancreatic cancer, stomach cancer, liver cancer, breast cancer, cervical cancer, Thyroid cancer, parathyroid cancer, lung cancer, non-small cell lung cancer, prostate cancer, gallbladder cancer, biliary tract cancer, non-Hodgkin lymphoma, Hodgkin lymphoma, blood cancer, bladder cancer, kidney cancer, melanoma, colon cancer, bone cancer, skin cancer, head cancer, uterine cancer, Rectal cancer, perianal cancer, fallopian tube carcinoma, endometrial carcinoma, vaginal cancer, vulvar carcinoma, esophageal cancer, small intestine cancer, endocrine cancer, adrenal cancer, soft tissue sarcoma, urethral cancer, penile cancer, ureteral carcinoma, renal cell carcinoma, renal pelvic carcinoma, central It may be a CNS central nervous system tumor, primary CNS lymphoma, or spinal cord tumor, preferably brain cancer, more preferably glioma or glioblastoma, but is not limited thereto.

In the present invention, the “cancer origin cell” refers to an origin cell with a mutation that causes cancer, and for the purpose of the present invention, the cancer origin cell may be a brain cancer origin cell.

In the present invention, the “brain cancer origin cell” may be a cell of the subventricular region or a cell derived therefrom, and may be a cell that migrated from the subventricular region, and is preferably an astrocyte ribbon cell or a cell thereof in the subventricular region. It may be a derived cell, and more preferably, it may be astrocyte-like neural stem cells of the astrocyte zone of the subventricular region or cells derived therefrom.

In the present invention, the “subventricular zone” refers to an area located on the lateral wall of the lateral ventricle, almost in contact with the ependymal layer. Many proliferating cells are concentrated in the subventricular zone. Rather, it has the characteristic of being composed of various types of nervous system cells at various stages of maturation.

More specifically, in the present invention, the brain cancer origin cell may share at least one mutation among TERT 1,295,228 C>T (C228T) and TERT 1,295,250 C>T (C250T) with the brain cancer cell, and the mutation measured in the brain cancer origin cell The expression level (frequency of variant allele) of at least one mutation among TERT 1,295,228 C>T (C228T) and TERT 1,295,250 C>T (C250T) was measured in the brain cancer cells. TERT 1,295,228 C>T (C228T) and TERT may be lower than the expression level (frequency of variant allele) of at least one mutation among 1,295,250 C>T (C250T).

In addition, in the present invention, the brain cancer origin cells are from the group consisting of brain cancer cells and EGFR (Epidermal growth factor receptor) mutation, TP53 (Tumor protein p53) mutation, PTEN (Phosphatase and tensin homolog) mutation, and Rb1 (Retinoblastoma 1) mutation. may share at least one selected mutation, and the frequency or copy number variation of the variant allele of the mutation measured in the brain cancer origin cell is smaller than the frequency or copy number variation of the variant allele measured in the brain cancer cell. You can.

The composition of the present invention can further improve the anticancer effect by further including commonly used anticancer agents. Here, the anticancer drugs include nitrogen mustard, imatinib, oxaliplatin, rituximab, erlotinib, neratinib, lapatinib, gefitinib, vandetanib, nirotinib, semasanib, bosutinib, axitinib, and masitib. nib, cediranib, lestaurtinib, trastuzumab, gefitinib, bortezomib, sunitinib, pazopanib, toceranib, nintedanib, regorafenib, semaxanib, tivozanib, ponatinib , cabozantinib, carboplatin, sorafenib, lenvatinib, bevacizumab, cisplatin, cetuximab, Viscum album, asparaginase, tretinoin, hydroxycarbamide, dasatinib, estramustine, gemtuzumab Ozogamycin, ibritumomabtuxetan, heptaplatin, methylaminolevulinic acid, amsacrine, alemtuzumab, procarbazine, alprostadil, holmium nitrate chitosan, gemcitabine, doxyfluridine, pemetrec. Ced, tegafur, capecitabine, gimeracin, oteracil, azacitidine, methotrexate, uracil, cytarabine, 5-fluorouracil, fludagabine, enocitabine, flutamide, capecitabine, Decitabine, mercaptopurine, thioguanine, cladribine, carmophor, raltitrexed, docetaxel, paclitaxel, irinotecan, belotecan, topotecan, vinorelbine, etoposide, vincristine, vinblastine, teniposide , doxorubicin, idarubicin, epirubicin, mitoxantrone, mitomycin, bleromycin, daunorubicin, dactinomycin, pyrarubicin, aclarubicin, pepromycin, temsirolimus, temozolomide. , busulfan, ifosfamide, cyclophosphamide, melphalan, altretmin, dacarbazine, thiotepa, nimustine, chlorambucil, mitolactol, leucovorin, tretonin, exemestane, aminoglute. Simide, anagrelide, olaparib, navelvin, padrazole, tamoxifen, toremifene, testolactone, anastrozole, letrozole, borozole, bicalutamide, lomustine, vorinostet, entinostat. and carmustine, but is not limited thereto.

In the present invention, “prevention” may include without limitation any act of blocking, suppressing or delaying cancer symptoms using the pharmaceutical composition of the present invention.

In the present invention, the pharmaceutical composition may be in the form of a capsule, tablet, granule, injection, ointment, powder, or beverage, and the pharmaceutical composition may be intended for human subjects.

The pharmaceutical composition of the present invention is not limited to these, but can be formulated and used in the form of oral dosage forms such as powders, granules, capsules, tablets, and aqueous suspensions, external preparations, suppositories, and sterile injection solutions according to conventional methods. You can.

The pharmaceutical composition of the present invention may include a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include binders, lubricants, disintegrants, excipients, solubilizers, dispersants, stabilizers, suspending agents, colorants, flavorings, etc. for oral administration, and buffers, preservatives, and analgesics for injections. Topics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used, and for topical administration, bases, excipients, lubricants, preservatives, etc. can be used.

The dosage form of the pharmaceutical composition of the present invention can be prepared in various ways by mixing it with a pharmaceutically acceptable carrier as described above. For example, for oral administration, it can be manufactured in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and for injections, it can be manufactured in the form of unit dosage ampoules or multiple dosage forms. there is. In addition, it can be formulated as a solution, suspension, tablet, capsule, sustained-release preparation, etc.

Meanwhile, examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, gum acacia, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, or mineral oil may be used. In addition, fillers, anti-coagulants, lubricants, wetting agents, fragrances, emulsifiers, preservatives, etc. may be additionally included.

The route of administration of the pharmaceutical composition according to the present invention is not limited to these, but is oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, and topical. , sublingual or rectal. Oral or parenteral administration is preferred.

As used herein, “parenteral” includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques. The pharmaceutical composition of the present invention can also be administered in the form of a suppository for rectal administration.

The pharmaceutical composition of the present invention may vary depending on several factors, including the activity of the specific compound used, age, body weight, general health, gender, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated. It may vary, and the dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, degree of disease, drug form, administration route, and period, but may be appropriately selected by a person skilled in the art, and may be administered at 0.0001 to 50 mg/day. It can be administered in kg or 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered several times. The above dosage does not limit the scope of the present invention in any way. The pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.

The pharmaceutical composition of the present invention can not only effectively improve or treat cancer by specifically killing cancer origin cells, but can also prevent the onset of cancer itself or prevent cancer recurrence.

Figure 1 shows perphenazine (P) (10 uM, 25 uM, 50 uM) and 2-deoxy-D-glucose (2DG) in subventricular zone cells (SVZ), the cells of origin of glioblastoma in Example 1. A graph shows the change in cell viability after treatment with a combination of 1 mM) and metformin (Met) (1 mM).
Figure 2 shows WST measuring changes in cell viability after treating perphenazine, 2-deoxy-D-glucose, and metformin in combination at different concentrations to subventricular zone cells (SVZ), which are the origin cells of glioblastoma, in Example 2. A dose-response matrix is shown based on the assay results.
Figure 3 shows WST measuring changes in cell viability after treating perphenazine, 2-deoxy-D-glucose, and metformin in combination at different concentrations to subventricular zone cells (SVZ), which are the origin cells of glioblastoma, in Example 2. This shows the results of measuring the Bliss synergy score based on the assay results.

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

[Preparation Example 1] Preparation of cancer origin cells

A mouse glioblastoma model was constructed in the same manner as in Korean Patent Publication No. 10-2019-0095074. Specifically, LoxP-Stop-LoxP EGFRvii f/+;LoxP-Stop-LoxP tdTomato f/ obtained by crossing LoxP-Stop-LoxP EGFRviii mice (FVB strain) and LoxP-Stop-LoxP-tdTomato mice (C57BL/6). + In mice, protein 53 (P53) and phosphatase and tensin homolog (PTEN) genes are selectively removed or mutated using CRISPR-Cas9 technology to create a mouse glioblastoma model. was built. After 14 weeks of mouse model construction, the mice were euthanized and their brains were removed. From the extracted mouse brain, the subventricular zone (SVZ), which corresponds to the origin of glioblastoma, and the caudal cortex, where cells migrated from the subventricular zone, were separated. MEM/nutrient mixture F-12 (DMEM/F-12; Mediatech) was added to the subventricular zone (SVZ) tissue and the caudal cortex tissue where cells migrated from the subventricular zone, respectively, and then separated using a scalpel. I ordered it. Afterwards, the separated samples were passed through a 100-um nylon mesh cell strainer (BD Falcon, Franklin Lakes, NJ, USA). The cell suspension was washed twice in DEME/F-12 and supplemented with 1 x B27 complement (Invitrogen, San Diego, CA, USA), 20 ng/ml basic fibroblast growth factor (bFGF; Sigma, St. Louis, MO, USA). ), 20 ng/ml epidermal growth factor (EGF; Sigma), 50 U/ml penicillin, and 50 mg/ml streptomycin.

[Example 1] Synergistic effect of perphenazine, metformin, and 2-deoxy-D-glucose on killing cancer cells (1)

Subventricular zone cells (SVZ), the origin cells of glioblastoma, were inoculated into a 96 well plate at 10,000 cells/well, and after 24 hours of culture, perphenazine, metformin, and 2-deoxy -D-glucose (2-deoxyglucose) was added to the medium treated with different concentrations. Then, after culturing for 72 hours, WST reagent (D-Plus ^TM CCK cell viability assay kit, Dongin LS) was added and cultured for an additional 2 hours. Absorbance was measured at 450 nm to calculate the % compared to the control to determine the treatment of each group. Cell survival rate was measured. The results are shown in Figure 1.

As shown in Figure 1, compared to the case where subventricular zone cells, which are carcinogenic cells of glioblastoma, are treated in combination with metformin and 2-deoxy-D-glucose, the cell survival rate is significantly reduced when treated in combination with perphenazine. I was able to confirm that

[Example 2] Synergistic effect of perphenazine, metformin, and 2-deoxy-D-glucose on killing cancer cells (2)

Based on the results of the WST assay on subventricular zone cells (SVZ), which are the origin cells of glioblastoma, performed in Example 1, perphenazine, The combined effect of three drugs, metformin and 2-deoxy-D-glucose, on the death of cancer cells was predicted, and the results are shown in Figures 2 and 3. However, the Bliss synergy score below was performed based on the reference (Aleksandr et al. SynergyFinder: a web application for analyzing drug combination dose-response matrix data. Bioinformatics, 33, 15, 2017, 2413-2415) It has been done.

By applying the concentration combination of each drug used in combination as 4x4, the larger the difference between the value predicted by the Bliss model and the actual value, the higher the payoff will be. As shown in Figures 2 and 3, the combination of perphenazine, metformin, and 2-deoxy-D-glucose showed a generally positive red color at each concentration, and the Bliss synergy score was calculated to be 14.433. It was confirmed that the combination of these three drugs had a synergistic effect on the death of cancer cells.

Although the embodiments of the present invention have been described in detail above, the scope of the present invention is not limited thereto, and various modifications and variations are possible without departing from the technical spirit of the present invention as set forth in the claims. This will be self-evident to those with ordinary knowledge in the field.

Claims (11)

(1) perphenazine or a pharmaceutically acceptable salt thereof;
(2) metformin or a pharmaceutically acceptable salt thereof; and
(3) A pharmaceutical composition for the prevention or treatment of brain cancer containing 2-deoxy-D-glucose or a pharmaceutically acceptable salt thereof as an active ingredient. According to paragraph 1,
The composition is a pharmaceutical composition for specifically killing cells of origin of brain cancer. delete delete delete According to paragraph 2,
A pharmaceutical composition, wherein the cell of origin of the brain cancer is a subventricular zone (SVZ) cell or a cell that migrated from the subventricular zone. According to paragraph 1,
The pharmaceutical composition is for preventing the development of brain cancer or recurrence of brain cancer. delete delete delete delete