Cancer treatment using brms

Abstract

The present disclosure provides a cancer treatment that uses BRMs. A specific embodiment provides a cancer treatment that uses a combination of BRMs and an ascorbic acid component. The present disclosure provides a use, method, combination, composition, or the like for cancer treatment. According to one embodiment, the cancer may be breast cancer (including triple-negative breast cancer, which lacks estrogen receptors, progesterone receptors, and HER2). The subject to be treated can be a mammal such as a human, a mouse, a rat, a cat, or a dog.

Description

Cancer treatment with BRMs

[0001] This disclosure relates to the treatment of cancer using BRMs (Biological Response Modifiers). More specifically, this disclosure relates to the treatment of cancer using a combination of BRMs and ascorbic acid components.

[0002] Cell-based medical treatments, such as regenerative medicine using iPS cells and CAR-T therapy, are being developed. However, cell-based therapies have problems, such as the risk of cancer.

[0003] The inventors of this invention have discovered that BRMs are useful in the treatment of cancer, and have completed this invention.

[0004] This disclosure provides, for example, the following items: (Item 1) A combination for treating cancer comprising BRMs and an ascorbic acid component. (Item 2) Any combination of the above items, wherein the cancer comprises breast cancer or cervical cancer. (Item 3) Any combination of the above items, wherein the cancer comprises breast cancer. (Item 4) Any combination of the above items, wherein the cancer comprises triple-negative breast cancer. (Item 5) Any combination of the above items for treating human or non-human mammals. (Item 6) Any combination of the above items, comprising the ascorbic acid component in a ratio of about 3 to about 100 mg per 1 mL of BRMs. (Item 7) Any combination of the above items, wherein the ascorbic acid component is ascorbic acid or a derivative thereof or a salt thereof. (Item 8) Any combination of the above items, wherein the BRMs are obtained from the culture supernatant of mesenchymal stem cells. (Item 9) The BRMs are about 6 × 10 １０ A combination of any of the above items, comprising small extracellular vesicles (sEVs) of a certain size per mL or more. (Item 10) A composition for treating cancer, comprising BRMs, characterized in that it is used in combination with an ascorbic acid component. (Item 11) A composition for treating cancer, comprising an ascorbic acid component, characterized in that it is used in combination with BRMs.

[0005] This disclosure provides a novel cancer treatment.

[0006] This shows the effects of the combination of BRMs (ES) and ascorbic acid (AA) or reduced glutathione (GSH) on MCF-7 cells. The vertical axis shows the number of cells, and the horizontal axis shows the respective culture medium conditions on day 0 and day 6 of culture. These are microscopic images of MCF-7 cells cultured with the combination of BRMs (ES) and ascorbic acid (AA) or reduced glutathione (GSH). From top to bottom, the images are at 24 hours, 96 hours, and 120 hours from the start of culture. These are microscopic images of MCF-7 cells cultured with the combination of BRMs (ES) and ascorbic acid (AA) or reduced glutathione (GSH). From top to bottom, the images are at 24 hours, 96 hours, and 120 hours from the start of culture. This shows the effects of the combination of BRMs (ES) and ascorbic acid (AA) or reduced glutathione (GSH) on HeLa cells. The vertical axis shows the cell count, and the horizontal axis shows the culture medium conditions on day 0 and day 6 of culture. These are microscopic images of HeLa cells cultured with a combination of BRMs (ES) and ascorbic acid (AA) or reduced glutathione (GSH). From top to bottom, the images are from 24 hours, 96 hours, and 120 hours after the start of culture. These are microscopic images of HeLa cells cultured with a combination of BRMs (ES) and ascorbic acid (AA) or reduced glutathione (GSH). From top to bottom, the images are from 24 hours, 96 hours, and 120 hours after the start of culture. This shows the cell count on day 6 of MDA-MB468 cells (human triple-negative breast cancer) cultured with BRMs (ES) and ascorbic acid (AA). The vertical axis shows the relative cell count under each condition, with the cell count under the no-addition-drug condition (Control) set to 1. The horizontal axis shows the respective culture medium conditions. The cell count after culturing MCF-7 cells with BRMs (ES) and ascorbic acid derivatives for 5 days is shown. The vertical axis shows the cell count under each condition, and the horizontal axis shows the respective culture medium conditions.

[0007] The present invention will be described below with reference to illustrative examples, as necessary, with reference to the accompanying drawings. Throughout this specification, singular expressions should be understood to include the concept of their plural forms unless otherwise specified. Furthermore, terms used herein should be understood to have the meaning commonly used in the art unless otherwise specified. Thus, unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention pertains. In case of any conflict, this specification (including definitions) shall prevail.

[0008] (Definition) In this specification, “BRMs (Biological Response Modifiers)” means substances produced by cells that have some biological function, or compositions containing such substances. BRMs may be compositions containing substances produced by cells and released extracellularly during cell culture (sometimes abbreviated as “extracellular secretions” or “ES” in this specification). Typically, BRMs are preparations of the supernatant or components (extracellular vesicles, proteins, etc.) of a cell culture that does not contain cells themselves. BRMs are also obtained by removing specific components (extracellular vesicles, proteins, etc.) from the supernatant of a cell culture.

[0009] In this specification, “extracellular vesicles” (sometimes abbreviated as “EVs”) refer to vesicles smaller than cells that are located outside the cell and surrounded by a lipid membrane. Extracellular vesicles typically contain proteins (e.g., membrane proteins), nucleic acids, and lipids. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic bodies. Extracellular vesicles typically have a diameter of about 50 to 5000 nm. In this specification, sEVs refer to extracellular vesicles with a particle size of about 50 to 600 nm. The particle size of extracellular vesicles can be measured, for example, by nanotracking.

[0010] In this specification, “extracellular vesicles” (sometimes abbreviated as “EVs”) refer to vesicles smaller than cells that are located outside the cell and surrounded by a lipid membrane. Extracellular vesicles typically contain proteins (e.g., membrane proteins), nucleic acids, and lipids. Examples of extracellular vesicles include exosomes, microvesicles, and apoptotic bodies. Extracellular vesicles typically have a diameter of about 50 to 5000 nm. In this specification, sEVs refer to extracellular vesicles with a particle size of about 50 to 600 nm. The particle size of extracellular vesicles can be measured, for example, by nanotracking.

[0011] In this specification, “mesenchymal stem cells” are cells that have at least one of the following characteristics: (1) expression of specific cell membrane markers CD73, CD90, and CD105; (2) lack of expression of CD11b, CD14, CD34, CD45, CD19, CD79a, and HLA-DR; and (3) three germ layer pluripotency (the ability to differentiate into osteoblasts, chondrocytes, and adipocytes). Mesenchymal stem cells can be obtained from bone marrow, adipose tissue, teeth, etc., and can also be differentiated from pluripotent cells.

[0012] In this specification, “ascorbic acid component” refers to 3,4-dihydroxy-5-(1,2-dihydroxyethyl)furan-2(5H)-one (ascorbic acid) or its derivatives or salts thereof. The ascorbic acid component typically has the following structure It is a compound containing (also called vitamin C), but also includes its stereoisomers and racemic mixtures. Derivatives of ascorbic acid include ester derivatives and ether-linked derivatives. Derivatives of ascorbic acid may also be precursors of ascorbic acid that are metabolized in the bodies of mammals such as humans to produce ascorbic acid. Ester derivatives include saturated or unsaturated linear or branched fatty acids (C) such as ascorbyl palmitate or ascorbyl stearate. ２－２４ Examples include esters with (etc.), phosphate esters, etc. Examples of ether-bonded derivatives include ascorbic acid 2-glucoside, ethyl ascorbic acid, glyceryl ascorbic acid, and bisglyceryl ascorbic acid.

[0013] Derivatives of ascorbic acid may include, for example, compounds represented by the following formula: In the formula, the bond of the half-wave line represents a single bond or a double bond, and R １ is NR Ａ R Ｂ , OR Ａ or halogen, and R ２ is hydrogen, OR C or halogen, and R ３ and R ４ are each independently hydrogen, pentose, hexose, disaccharide (e.g., sucrose), C(=O)R Ｄ , or C ０－４ aliphatic chain - R Ｄ and R Ａ and R Ｂ are each independently hydrogen or R', or R Ａ and R Ｂ may together form a 5- to 10-membered monocyclic or bicyclic aromatic heterocyclic ring substituted with 0 to 4 substituents R', and R Ｃ is hydrogen or P(=O)(OR Ｘ ) ２ and R Ｘ are each independently hydrogen or (C ０－４ aliphatic chain)-phenyl, and R Ｄ are each independently hydrogen, a saturated or unsaturated straight-chain or branched C １－２４ aliphatic chain substituted with 0 to 4 substituents R'', a 5- to 6-membered alicyclic ring substituted with 0 to 4 substituents R'', or a 5- to 6-membered aromatic ring substituted with 0 to 4 substituents R'', and R' is each independently halogen, =O, a saturated or unsaturated straight-chain or branched C １－２４ aliphatic chain substituted with 0 to 4 substituents R'', or (C ０－４ aliphatic chain)-(5- to 6-membered aromatic ring) substituted with 0 to 4 substituents R'', and R'' is each independently a hydroxyl group, halogen, cyano group, C １－４ aliphatic chain, O-C １－４ aliphatic chain or O-(C ０－４It is (an aliphatic chain)-(a 5- or 6-membered aromatic ring).

[0014] Specific derivatives of ascorbic acid may include, for example, the following compounds: (Bn = benzyl, Ph = phenyl)

[0015] The phosphate ester of ascorbic acid is a compound in which one, two, three or four of the hydroxyl groups at the 2nd, 3rd, 5th and 6th positions of ascorbic acid form esters with phosphoric acid. Among ascorbic acid derivatives, it is expected that when combined with BRMs, a cancer-suppressing effect similar to that of ascorbic acid can be obtained. As the phosphate ester of ascorbic acid or its salt, ascorbyl phosphate Na (trisodium 2-phospho-L-ascorbate): and the like can be mentioned.

[0016] Examples of salts include, but are not limited to, salts with sodium, potassium, magnesium, calcium or ammonia.

[0017] In this specification, "treatment" includes both treatment and prevention. In this specification, "treatment" means, for a certain disease or disorder, when in such a state, preventing the deterioration of such a disease or disorder, preferably maintaining the current state, more preferably reducing it, and even more preferably eliminating it, and includes the ability to exert a symptom-improving effect or a preventive effect on the patient's disease or one or more symptoms associated with the disease. In this specification, "prevention" means, for a certain disease or disorder, preventing it from reaching such a state before reaching such a state.

[0018] In this specification, the term "about" refers to plus or minus 10% of the indicated value, unless otherwise defined. When "about" is used for temperature, it refers to plus or minus 5 °C of the indicated temperature, and when "about" is used for pH, it refers to plus or minus 0.5 of the indicated pH.

[0019] (Preferred Embodiment) The preferred embodiments of the present disclosure will be described below. The embodiments provided below are for a better understanding of the present disclosure, and it is understood that the scope of the present disclosure should not be limited to the following description. Therefore, it is clear that those skilled in the art can make appropriate modifications within the scope of the present disclosure by referring to the descriptions in this specification. Also, it is understood that the following embodiments of the present disclosure can be used alone or in combination with each other.

[0020] (Anti-cancer Use) The present disclosure provides a treatment for cancer using BRMs. In a specific embodiment, the present disclosure provides a treatment for cancer using a combination of BRMs and an ascorbic acid component. This specification will describe the present disclosure mainly centered on the description of the treatment for cancer using BRMs, but various aspects such as the use of BRMs in the treatment of cancer, the method of treating cancer including the step of administering BRMs, and the combination or composition for the treatment of cancer containing BRMs are also contemplated as inventions. The description of certain aspects such as the method is similarly applicable to other aspects.

[0021] In one embodiment, cancers to be treated include malignant and benign tumors (for example, primary, metastatic or recurrent breast cancer, prostate cancer, pancreatic cancer, stomach cancer, lung cancer, colorectal cancer (colon cancer, rectal cancer, anal cancer), esophageal cancer, duodenal cancer, head and neck cancer (tongue cancer, pharyngeal cancer, laryngeal cancer, thyroid cancer), brain tumors, schwannomas, neuroblastomas, gliomas, non-small cell lung cancer, small cell lung cancer, liver cancer, kidney cancer, bile duct cancer, uterine cancers (endometrial cancer, cervical cancer), ovarian cancer, bladder cancer, skin cancer, hemangiomas, malignant lymphomas, malignant melanomas, bone tumors, angiofibromas, retinal sarcomas, penile cancers, pediatric solid tumors, Kaposi's sarcoma, maxillary sinus tumors, fibrous histiocytoma, leiomyosarcoma, rhabdomyosarcoma, liposarcoma, uterine fibroids, osteoblastoma, osteosarcoma, chondrosarcoma, malignant mesothelioma, leukemia, and other tumors). In one embodiment, the cancer may be breast cancer (including triple-negative breast cancer lacking estrogen receptor, progesterone receptor, and HER2). In one embodiment, the cancer to be treated is characterized by elevated Stat3 expression and includes cancer cells exhibiting Stat3 expression levels (e.g., mRNA count, protein count) about 1.2 times, about 1.5 times, about 2 times, about 3 times, about 4 times, about 5 times, about 7 times, about 10 times, or greater than the average Stat3 expression levels (e.g., mRNA count, protein count) in normal cells in the organ with cancer or in cells in the same organ of a healthy subject without cancer.

[0022] Examples of animals that can be treated include mammals such as humans, mice, rats, cats, and dogs. In one embodiment, the treated animal is a human. In another embodiment, the treated animal is a pet (dog, cat, etc.). In another embodiment, BRMs derived from cells of the same species as the treated animal may be used.

[0023] (Dosage and Administration) In one embodiment, BRMs are administered to a subject in doses ranging from approximately 5 to approximately 100 mL, for example, approximately 5, approximately 10, approximately 15, approximately 20, approximately 30, approximately 40, approximately 50, approximately 60, approximately 70, approximately 80, approximately 90, approximately 100 mL, or a range between two of these values. In a typical embodiment, BRMs are administered to a subject in doses ranging from approximately 30 mL. In one embodiment, BRMs are administered to a subject in doses ranging from approximately 0.1 to approximately 2 mL per body weight, for example, approximately 0.1, approximately 0.2, approximately 0.3, approximately 0.4, approximately 0.5, approximately 0.6, approximately 0.7, approximately 0.8, approximately 0.9, approximately 1, approximately 1.2, approximately 1.5, approximately 1.7, approximately 2 mL, or a range between two of these values.

[0024] In one embodiment, the ascorbic acid component is administered to a subject in doses ranging from about 3 to about 300 g at a time, for example, about 3, about 5, about 7, about 10, about 15, about 20, about 30, about 40, about 50, about 70, about 100, about 150, about 200, about 250, about 300 g, or within a range between two of these values. In one embodiment, the ascorbic acid component is administered to a subject in doses ranging from about 50 to about 5000 mg per body weight at a time, for example, about 50, about 70, about 100, about 150, about 200, about 300, about 400, about 500, about 700, about 1000, about 1500, about 2000, about 3000, about 4000, about 5000 mg, or within a range between two of these values. In a typical embodiment, the ascorbic acid component is administered by intravenous infusion at a dose of approximately 100 to 1000 mg per body weight of the subject.

[0025] In one embodiment, BRMs and ascorbic acid components are administered in a proportion of about 1 to about 200 mg of ascorbic acid component per 1 mL of BRMs, for example, about 1, about 2, about 3, about 4, about 5, about 7, about 10, about 15, about 20, about 30, about 40, about 50, about 70, about 100, about 120, about 150, about 200 mg, or a range between two of these values. For ascorbic acid components that are ascorbic acid derivatives and / or salts, the above dosage may be calculated based on the amount of ascorbic acid contained therein or derived therefrom.

[0026] The amounts of BRMs and ascorbic acid components may vary depending on the target of treatment or the nature of the cancer, but those skilled in the art can determine them by standard clinical techniques based on the description herein. In some cases, in vitro assays may be used to assist in identifying the optimal dosage range. The exact dose to be used in the formulation may also vary depending on the route of administration and the severity of the disease or condition, and should be determined according to the judgment of the attending physician and the circumstances of each patient. The dosing interval is not particularly limited, but may be administered once or twice every 1, 7, 14, 21, or 28 days, or once or twice every two of these intervals. The dosage, dosing interval, and method of administration may be appropriately selected depending on the age, weight, symptoms, etc. of the subject.

[0027] The administration routes for BRMs and ascorbic acid components may include, for example, intravenous (e.g., intravenous infusion), intradermal, subcutaneous, intramuscular, intraperitoneal, intrathecal, intraventricular, intraparenchymal, and epidural administration. Local administration is also possible, but even localized cancers can be treated by intravenous administration.

[0028] BRMs and ascorbic acid may be administered simultaneously or at different times. When BRMs and ascorbic acid are administered simultaneously, they may be administered mixed or separately.

[0029] In addition to treatment with BRMs and ascorbic acid components, further treatment with other drugs and / or therapies may be performed. For example, typically, other drugs and / or therapies for the treatment of cancer or its complications may be combined.

[0030] (BRMs) In one embodiment, BRMs contain the culture supernatant or components of mesenchymal stem cells. Therefore, BRMs may contain the culture medium components used to culture the cells (e.g., salts, buffers, growth factors, vitamins, minerals, cytokines, etc.). In one embodiment, BRMs do not contain cells. Cells can be easily removed from the culture supernatant using a filter or the like. The BRMs of the present invention have been shown to improve mitochondrial activity (data not shown), and while not intended to be theoretically bound, it is possible that this improvement in mitochondrial activity leads to the improved cancer-suppressing effect when combined with ascorbic acid.

[0031] In one embodiment, the BRMs of the present invention do not contain extracellular vesicles. In one embodiment, the BRMs of the present invention further contain extracellular vesicles.

[0032] In one embodiment, BRMs are characterized by small extracellular vesicles (sEVs). Excessive mTOR activation can be suppressed by small extracellular vesicles (sEVs). In one embodiment, BRMs are approximately 1 × 10⁻⁶ １０ pcs / mL or more, approximately 2 x 10 １０ pcs / mL or more, approximately 5 x 10 １０ pcs / mL or more, approximately 5 x 10 １０ pcs / mL or more, approximately 1×10 １１ More than 5 pieces / mL or approximately 5 x 10 １１ More than 5 pieces / mL and / or about 5 × 10 １０ pcs / mL or less, approximately 1×10 １１ pcs / mL or less, approximately 2 x 10 １１ pcs / mL or less, approximately 5 x 10 １１ Less than or equal to 1 x 10 cells / mL １２ Contains small extracellular vesicles (sEVs) of less than 1 / mL.

[0033] In one embodiment, the small extracellular vesicles (sEVs) in the BRMs have a mode particle size of approximately 90–130 nm, 90–120 nm, 90–115 nm, 90–110 nm, 100–130 nm, 100–120 nm, 100–115 nm, 100–110 nm, 105–130 nm, 105–120 nm, or 105–115 nm.

[0034] In one embodiment, the small extracellular vesicles (sEVs) in the BRMs have a particle size distribution with a full width at half maximum of approximately 40–90 nm, 50–80 nm, 55–75 nm, or 60–70 nm.

[0035] In one embodiment, the extracellular vesicles in the BRMs of the present invention may have a proportion of small-sized extracellular vesicles (sEVs) of about 80% or more, about 85% or more, about 90% or more, about 95% or more, about 97% or more, or about 99% or more of the total extracellular vesicles.

[0036] (Preparation of BRMs) In one embodiment, BRMs can be prepared from the culture supernatant of mesenchymal stem cells. Specifically, BRMs may be the culture supernatant obtained by removing the cells themselves and cell debris from the culture medium of mesenchymal stem cells. Although not intended to be constrained by theory, many tumors are GLUT1 high-expressing, and ascorbic acid is thought to be more easily taken up into cells than glutathione, consuming NADPH and other substances within the cell and depleting redox buffering capacity. On the other hand, the BRMs of the present invention are expected to promote the depletion of redox buffering capacity by resulting in improved mitochondrial activity. Since improved mitochondrial activity is a characteristic observed not only in adipose-derived mesenchymal stem cells but also in mesenchymal stem cells of other origins (e.g., dental pulp cells, bone marrow cells, umbilical cord cells, umbilical cord blood, iPS cells), it is thought that the cancer-suppressing effect when combined with ascorbic acid can be achieved not only in adipose-derived BRMs but also in BRMs prepared from the culture supernatant of mesenchymal stem cells of other origins. In one embodiment, the mesenchymal stem cells are human cells. In one embodiment, mesenchymal stem cells are derived from adipocytes, dental pulp cells, bone marrow cells, umbilical cord cells, umbilical cord blood, nerve cells, epithelial cells, endothelial cells, hematopoietic cells, connective tissue cells, muscle cells, osteocytes, chondrocytes, germline cells, or iPS cells. In a preferred embodiment, mesenchymal stem cells are derived from adipocytes. In a preferred embodiment, mesenchymal stem cells are derived from dental pulp cells. In a preferred embodiment, mesenchymal stem cells are derived from bone marrow cells. In a preferred embodiment, mesenchymal stem cells are derived from umbilical cord cells. In a preferred embodiment, mesenchymal stem cells are derived from umbilical cord blood. In a preferred embodiment, mesenchymal stem cells are derived from human iPS cells.

[0037] Cell culture conditions can affect the components of BRMs (such as extracellular vesicles). In one embodiment, the BRMs of this disclosure are prepared from the culture supernatant of mesenchymal stem cells cultured at pH levels of approximately 7.0 to 10.0, 7.0 to 9.5, 7.0 to 9.0, 7.0 to 8.5, 7.5 to 10.0, 7.5 to 9.5, 7.5 to 9.0, 7.5 to 8.5, 8.0 to 10.0, 8.0 to 9.5, 8.0 to 9.0, 8.0 to 8.5, 8.5 to 10.0, or 8.5 to 9.5. In one embodiment, the culture medium for mesenchymal stem cells comprises Tris hydrochloride buffer, HEPES buffer, or sodium bicarbonate buffer, preferably sodium bicarbonate buffer. In one embodiment, the culture medium for mesenchymal stem cells contains magnesium (for example, about 0.001 mM to about 1 mM, e.g., about 0.001 mM, about 0.01 mM, about 0.1 mM, about 1 mM, etc.).

[0038] To prepare BRMs from cell culture supernatant, operations such as integration and distribution of the collected culture supernatant, freezing, and filtering can be performed.

[0039] In one embodiment, cell culture supernatant can be separated into an extracellular vesicle fraction and a supernatant fraction. The fraction containing a higher concentration of extracellular vesicles compared to the original cell culture supernatant is called the extracellular vesicle fraction, and the fraction containing a lower concentration of extracellular vesicles is called the supernatant fraction. Methods such as centrifugation, sucrose density gradient ultracentrifugation, tangential flow filtration, and size exclusion chromatography can be used for separation. The BRMs of this disclosure may be provided as a combination of the extracellular vesicle fraction and the supernatant fraction. In one embodiment, the extracellular vesicle fraction contains extracellular vesicles or small-sized extracellular vesicles (sEVs) at concentrations of at least about 110%, at least about 120%, at least about 150%, at least about 200%, at least about 250%, at least about 300%, at least about 500%, at least about 700%, at least about 1000%, at least about 1500%, or at least about 2000% of the cell culture supernatant. In one embodiment, the supernatant fraction contains extracellular vesicles or small-sized extracellular vesicles (sEVs) at concentrations of up to approximately 90%, up to approximately 80%, up to approximately 50%, up to approximately 20%, up to approximately 15%, up to approximately 10%, up to approximately 7%, up to approximately 5%, up to approximately 2%, up to approximately 1.5%, or up to approximately 1% of the cell culture supernatant.

[0040] In a typical embodiment, the BRMs of this disclosure are not exosome concentrates such as purified exosome preparations, but rather liquids that remove cells, extracellular matrix and their fragments from a cell culture, while retaining most of the other components of the cell culture (including extracellular vesicles) in an amount equivalent to that of the original cell culture. In a particular embodiment, the BRMs of this disclosure are supernatant fractions prepared by removing at least some extracellular vesicles from a cell culture supernatant (e.g., prepared by removing cells from a cell culture). In one embodiment, the extracellular vesicles contained in the BRMs amount to about 1 × 10⁶ ６ pcs / mL or less, approximately 1×10 ５ pcs / mL or less, approximately 1×10 ４ pcs / mL or less, approximately 1×10 ３ Less than or equal to 1 x 10 cells / mL ２ The number of particles per mL is less than or equal to 1 / mL.

[0041] (Combinations or Formulations) In one embodiment, the Disclosure provides combinations or compositions comprising the BRMs and / or ascorbic acid components of the Disclosure. Each element constituting the combination may be a composition. The composition may include, for example, isotonic agents, thickeners, sugars, sugar alcohols, preservatives, bactericides, pH adjusters, stabilizers, chelating agents, oily bases, gel bases, surfactants, suspending agents, binders, excipients, lubricants, disintegrants, foaming agents, fluidizers, dispersants, emulsifiers, buffers, solubilizers, antioxidants, and the like.

[0042] The combinations or compositions described herein may be provided in various forms. Compositions containing BRMs and / or ascorbic acid components may be in the form of injections, reconstituted powders (e.g., lyophilized agents), etc., but are preferably in the form of solutions. Aqueous solutions for injection may be stored, for example, in vials or stainless steel containers. The aqueous solutions for injection may also contain, for example, physiological saline, sugar (e.g., trehalose), NaCl, or NaOH. In preferred embodiments, the compositions can be formulated as pharmaceutical compositions adapted for administration to humans according to known methods. Such compositions can be administered by injection. Typically, compositions for injection are solutions in sterile isotonic aqueous buffer. If necessary, the composition may also include solubilizers and local anesthetics such as lidocaine to relieve pain at the injection site. Generally, the components may be supplied separately or mixed together in unit dose forms, and can be supplied as lyophilized powders or water-free concentrates in sealed containers such as ampoules or sachets indicating the amount of the activator. When administering the composition by injection, it is also possible to dispense it using an injection bottle containing sterile-grade water or saline solution. When administering the composition by injection, it is also possible to provide an ampoule of sterile water or saline solution for injection so that the components can be mixed before administration. The composition may have a pH of, for example, about 5.0 to 9.0, about 5.5 to 8.5, or about 6.0 to 8.0.

[0043] Combinations or compositions of the present disclosure may be provided as kits. In one embodiment, the present disclosure provides a drug pack or kit comprising one or more containers filled with one or more components that may be added to the compositions of the present disclosure. Optionally, such containers may also include information indicating authorization by a government agency for manufacture, use or sale for human administration, in a form prescribed by the government agency that regulates the manufacture, use or sale of pharmaceutical or biological products.

[0044] (Other Embodiments) The present invention has been described above with reference to preferred embodiments for ease of understanding. The present invention will now be described based on examples, but the above description and the following examples are provided for illustrative purposes only and are not intended to limit the present invention. Accordingly, the scope of the present invention is not limited to the embodiments or examples specifically described herein, but is limited only by the claims.

[0045] (Example 1: Preparation of BRMs) Adipose-derived mesenchymal stem cells were thawed, and after centrifugation (800 rpm, 5 minutes), the supernatant was discarded. Then, fresh cell culture medium was added, and the cells were cultured. The pH was adjusted to approximately 8.0 with sodium bicarbonate, and the culture supernatant was obtained after removing the cells themselves and cell debris, and this was designated as BRMs. In the following examples, unless otherwise specified, BRMs refer to the entire culture supernatant from which extracellular vesicles have not been separated.

[0046] Using a NanoSight NS300 (Malvern, UK) CMOS camera, a violet laser (405 nm, <65 mW), and analysis software: NTA3.4, the size of EVs in BRMs was measured, and it was found that the BRMs contained approximately 6.15 × 10⁻¹⁶ EVs. １０ Total EVs per mL: approximately 6.1 × 10⁻⁶ １０The culture supernatant contained sEVs (with particle sizes of 50-600 nm) at a concentration of 1 / mL, with a mode particle size of approximately 108 nm and a full width at half maximum of approximately 66 nm. The majority of the EVs were sEVs. The culture supernatant can be separated into extracellular vesicles (EVs) and the supernatant by centrifugation. In another experiment, it was confirmed that the supernatant of BRMs acts as an accelerator, activating the mTOR and MAPK pathways, while EVs act as a brake, suppressing the activated mTOR and MAPK pathways to some extent. It has also been confirmed that BRMs can reduce cellular senescence. In addition, tests were conducted on cultured cells separately to investigate the basic effects of BRMs. For example, when HeLa cells (derived from human cervical cancer cells) were treated with BRMs, it was confirmed using JC-1 that BRMs can activate mitochondria.

[0047] (Example 2: Cancer treatment using a combination of BRMs and antioxidants) Based on the findings from treatments using BRMs, it was considered possible that a combination of BRMs and antioxidants could effectively treat cancer, so a test was conducted. Ascorbic acid and reduced glutathione were tested as antioxidants.

[0048] (Materials and experimental procedure) Cell culture MCF-7 cells (human mammary gland cancer cells) and HeLa cells (human cervical cancer cells) were cultured in Dulbecco's modified Eagle medium containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin at 5% CO2. ２ The cultured cells were incubated at 37°C. The cultured cells were placed in a 24-well plate at a rate of 1 x 10⁶. ４ The seeds were seeded to form a cell-to-well ratio.

[0049] • Preparation of test media: L-ascorbic acid (Sigma A5960) was dissolved in water at a concentration of 500 mmol / L and filtered and sterilized through a 0.22 μm filter. Reduced glutathione (Sigma G6013) was dissolved in water at a concentration of 150 mmol / L and filtered and sterilized through a 0.22 μm filter. BRMs were added to Dulbecco's modified Eagle medium containing 10% fetal bovine serum (FBS) and 1% penicillin streptomycin to a final concentration of 5%, L-ascorbic acid to a final concentration of 1 mmol / L, and reduced glutathione to a final concentration of 10 mmol / L. The day after cell seeding, the medium was replaced with this added test medium and incubated in this state for 5 days (120 hours) under 5% CO2. ２ They were cultured at 37°C.

[0050] Five days after replacing the culture medium with the cell counting test medium, the cell count was measured. The culture medium in the 24-well plate was removed, washed with PBS, and the cells were detached from the wells with trypsin / EDTA solution and collected. The collected cells were collected by centrifugation, suspended in culture medium, and the cell count was measured using a hemocytometer.

[0051] Cell observation and photography: Each cell was observed morphologically and photographed using a KEYENCE inverted fluorescence phase-contrast microscope BZ-X710. A 10x lens was used.

[0052] (Results) The test results for MCF-7 cells are shown in Figures 1-3, and the test results for HeLa cells are shown in Figures 4-6. Compared to the control, BRMs (ES) alone showed an inhibitory effect on both cancer cells, but the cancer-suppressing effect was further enhanced by combining them with ascorbic acid (AA). On the other hand, the antioxidant reduced glutathione (GSH) did not enhance the cancer-suppressing effect when added to BRMs (ES). From this, it is considered that the combination of BRMs and ascorbic acid is particularly favorable for cancer suppression.

[0053] (Example 3: Treatment of Triple-Negative Breast Cancer) In the above example, we confirmed that the combination of BRMs and ascorbic acid is effective in suppressing breast cancer. Next, we tested its effect on triple-negative breast cancer, which is particularly aggressive.

[0054] MDA-MB-468, a well-known cell model for human triple-negative breast cancer, was passed through two generations and then plated in 24-well plates in a 1x10⁶ format. ４ Cells were seeded to a cell / well ratio. The day after seeding, BRMs were added to a final concentration of 5% and L-ascorbic acid to a final concentration of 100 μmol / L. Cells were cultured for 6 days and then observed. The following four conditions were tested: control condition with no additional drugs (Control), BRMs only (ES), ascorbic acid only (AA 100 μM), and a combination of BRMs and ascorbic acid (ES + AA).

[0055] (Results) Figure 7 shows a comparison of cell counts on day 6. The vertical axis shows the relative cell count in each condition, with the cell count under the no-drug condition (Control) set to 1. When BRMs alone (ES) and ascorbic acid alone (AA 100uM) were added, the cell count increased compared to Control. However, when a combination of BRMs and ascorbic acid (ES + AA) was added, a decrease in cell count was observed compared to the control condition (Control) without additional drug addition. From this, it is thought that a synergistic cancer-suppressing effect can be obtained when a combination of BRMs and ascorbic acid is added even in triple-negative breast cancer cells.

[0056] (Example 4: Effects on Stat3) The effects of BRMs on Stat3, which is known to be associated with cancer, were investigated.

[0057] MCF-7 cells (known to have elevated Stat3 expression) were cultured in the same manner as in Example 2, and BRMs were added in the same manner as in Example 2. Three days after seeding, the cells were harvested, and Western blotting was performed for Stat3.

[0058] As a result, it was found that the expression level of Stat3 protein was suppressed in cells treated with BRMs compared to untreated controls.

[0059] Stat3 is known to play an important role in the growth process of cancer, including promoting cell proliferation and suppressing apoptosis. It is also known that Stat3 activation is promoted by reactive oxygen species (ROS) (Autophagy. 2010 Nov;6(8):1125-38.). Ascorbic acid can remove ROS by reduction. Therefore, although we do not wish to be bound by any particular theory, it is conceivable that the combination of BRMs and ascorbic acid suppresses Stat3 by reducing both ROS and Stat3 protein levels, thereby exhibiting an inhibitory effect on cancer cell proliferation. For this reason, in addition to the specific examples shown above, the combination of BRMs and ascorbic acid disclosed herein may be particularly effective in treating cancers characterized by Stat3 activation.

[0060] (Example 4: Combination with ascorbic acid derivatives) We also tested whether a synergistic cancer-suppressing effect could be achieved by combining ascorbic acid derivatives with BRMs.

[0061] MCF-7 cells were cultured and BRMs were prepared in the same manner as in Example 2. Under the same culture conditions as in Example 2, MCF-7 cells (P7) were cultured for 5 days in a medium to which BRMs were added at a final concentration of 5% and sodium ascorbyl phosphate (2-phospho-L-ascorbate trisodium salt) was added from a final concentration of 1 mmol / L to a final concentration of 10 mmol / L, after which cell counting was performed.

[0062] (Results) Figure 8 shows the test results under the following conditions: no drug addition (Control), BRMs only (ES), sodium ascorbyl phosphate (ascorbic acid derivative) only, and BRMs + ascorbic acid derivative. Compared to the control, BRMs (ES) alone showed an inhibitory effect on both types of cancer cells, but the cancer-suppressing effect was further enhanced by combining them with sodium ascorbyl phosphate. Sodium ascorbyl phosphate alone did not show a strong cancer-suppressing effect. From this, it was confirmed that BRMs exert a strong cancer-suppressing effect when combined with ascorbic acid derivatives, similar to ascorbic acid.

[0063] This disclosure provides a novel cancer treatment.

Claims

1. A combination for treating cancer, containing BRMs and ascorbic acid components.

2. The combination according to claim 1, wherein the cancer includes breast cancer or cervical cancer.

3. The combination according to claim 1, wherein the cancer includes breast cancer.

4. The combination according to claim 1, wherein the cancer includes triple-negative breast cancer.

5. The combination according to claim 1 for treating a human or non-human mammal.

6. The combination according to claim 1, comprising the ascorbic acid component in a ratio of about 3 to about 100 mg per 1 mL of the BRMs.

7. The combination according to claim 1, wherein the ascorbic acid component is ascorbic acid or a derivative thereof or a salt thereof.

8. The combination according to claim 1, wherein the BRMs are obtained from the culture supernatant of mesenchymal stem cells.

9. The BRMs are approximately 6 × 10 １０ The combination according to claim 1, comprising small extracellular vesicles (sEVs) of a certain size per mL or more.

10. A composition for treating cancer, comprising BRMs, characterized in that it is used in combination with an ascorbic acid component.

11. A composition for treating cancer, comprising an ascorbic acid component, characterized in that it is used in combination with BRMs.

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