WO2022197063A1 - Anticancer adjuvant composition comprising tonsil-derived mesenchymal stem cell-controlling medium for promoting platelet production - Google Patents

Abstract

The present invention relates to an anticancer adjuvant pharmaceutical composition comprising a tonsil-derived mesenchymal stem cell-controlling medium. The composition of the present invention has marker and/or protein profiles different from those of mesenchymal stem cells derived from other sources and controlling media therefor and exhibits an excellent supplemental improvement effect on anticancer therapy and thus, can be used as an adjuvant for reducing platelets in anticancer therapy.

Description

Anti-cancer treatment adjuvant composition for promoting platelet production comprising a conditioned medium for tonsil-derived mesenchymal stem cells

The present invention relates to a method for preparing a T-MSC conditioned medium with increased expression of placental forming factor (PlGF), comprising culturing tonsil-derived mesenchymal stem cells, and/or to an adjuvant for promoting platelet production and anticancer treatment comprising the conditioned medium it's about

Cancer, the leading cause of death in Korea, is on the rise due to the aging population. Currently, surgery, chemotherapy, and radiation therapy are mainly used for chemotherapy.

In particular, in the case of chemotherapy and radiation therapy, except for surgery, various agents and radiation are used, but their application is limited to the range that the human body can tolerate. This is because the limited application of these chemotherapy and radiotherapy, no matter how excellent the efficacy in animal studies, offsets the effect in clinical practice, limits the scope of application, and limits the efficacy. This is due to the appearance of cancer cells with anticancer drug resistance or side effects due to the cytotoxicity of each therapy.

For example, cisplatin [cis-diamminedichloroplatinum; CDDP] is the most effective cancer treatment, and is one of the most useful drugs among about 30 anticancer drugs currently used in clinical practice, including testicular, ovarian, lung, head and neck cancer, bladder cancer, stomach cancer, It is known to have medicinal effects on cancers such as cervical cancer. However, recently, many cancers with resistance to cisplatin have been reported, and cancer cells with such resistance are currently a problem and there is a risk of recurrence after cancer treatment. Research on treatment by protein regulation is actively underway.

In addition, radiation therapy is an anti-cancer therapy that treats various cancers occurring in the human body by irradiating an appropriate amount of radiation to the affected area. have. Furthermore, there is a limit to radiation therapy because cancer cells acquire resistance due to repeated irradiation.

The side effects of chemotherapy and radiation therapy in such chemotherapy may deteriorate the quality of life of patients regardless of whether the chemotherapy is successful or not, and may act as another fatal etiology.

One of the fatal side effects is bone marrow suppression, which can lead to anemia due to decreased red blood cells, weakened immunity due to decreased white blood cells, and bleeding risk due to thrombocytopenia. Another side effect is a side effect on the liver and kidneys, which inhibits the decomposition and excretion of the anticancer drug in the liver, so that other side effects of the anticancer drug are severe in many cases. If the kidney function, which is an organ that excretes wastes in our body, is reduced by anticancer drugs, cancer or other factors, the excretion of anticancer drugs is delayed, and the vicious cycle of maximizing the side effects of anticancer drugs can be repeated, and symptoms of uremia caused by delayed excretion of wastes may appear. In addition, many side effects affecting the heart, reproductive organs, and nervous system according to the individual characteristics of anticancer chemotherapeutic agents or radiotherapy are known.

Therefore, in patients in need of chemotherapy, the management of the side effects is as important as chemotherapy itself, unlike other diseases. In addition to adjuvant treatments for general mild nausea and vomiting, these chemotherapy and radiotherapy, and cell therapy being developed recently, while enhancing the efficacy of cell therapy, minimize the side effects of chemotherapy, and serve as an adjuvant or enhancer that can be widely used There is no known anticancer therapeutic adjuvant comprising a biological composition for

Platelets, called thrombocytes, are colorless blood cells that help blood clot. The normal human platelet count ranges from 130,000 to 400,000 platelets per microliter (μl) of blood. As in the case of neutropenia, if the differentiation of HSCs into CMPs is inhibited, or if platelets are destroyed, for example as a result of an autoimmune disease, the concentration of platelets in the blood may drop below the standard range (thrombocytopenia) ). A platelet count of <50,000 platelets/μl (blood) is considered a serious condition, and a level of <20,000 platelets/μl (blood) can cause life-threatening internal bleeding spontaneously. Thrombocytopenia has few symptoms until the platelet count becomes extremely low, and impairment to clotting can include spontaneous bruising, very mild post-traumatic bruising, and petechia (small bleeding within the skin and mucous membranes). as evidenced by red or purple spots/blotches on the skin due to Other symptoms may include malaise (with or without bleeding), fatigue, and general weakness. Thrombocytopenia is caused by, for example, bacterial or viral infection, cirrhosis, chemotherapy or radiation therapy, acute leukemia, aplastic anemia, or autoimmune disease. It may be a side effect of various drugs. Like neutropenia, thrombocytopenia is a common side effect of chemotherapy or radiation therapy for cancer.

In particular, high-dose anticancer therapy causes serious side effects related to thrombocytopenia and the like.

High-dose chemotherapy is widely used as a standard treatment for malignant blood diseases and some solid cancers. During this process, high-dose chemotherapy used as a pretreatment causes a lot of damage not only to tumor cells but also to normal tissues. The main side effect, myelosuppressive effect, is more severe than that of the non-blood part.

On the other hand, tonsil-derived mesenchymal stem cell conditioned medium is located on the inside of the throat and the back of the nose to primarily defend our body from substances such as bacteria invading from the outside, and at the same time to protect the lymph and epithelial immunity. It refers to undifferentiated stem cells having the ability to differentiate into two or more new cells while having self-renewal ability derived from the amygdala, a tissue that functions as a tissue. With respect to tonsil-derived mesenchymal stem cells, there have been reports of therapeutic efficacy for skin inflammatory diseases, therapeutic efficacy for chronic inflammatory bowel disease, and the like.

[Prior art literature]

[Patent Literature]

(Patent Document 1) Republic of Korea Patent Publication No. 10-2016-0140451

The present invention provides a method for preparing a T-MSC conditioned medium with increased expression of placental forming factor (PlGF), comprising the step of culturing tonsil-derived mesenchymal stem cells in a medium containing low-dose DMEM.

The present invention provides a pharmaceutical composition for adjuvant anticancer treatment comprising a conditioned medium for tonsil-derived mesenchymal stem cells.

The present invention provides a pharmaceutical composition for preventing or treating megakaryocyte aplasia comprising a conditioned medium for tonsil-derived mesenchymal stem cells.

The present invention provides a pharmaceutical composition for preventing or treating thrombocytopenia comprising a conditioned medium for tonsil-derived mesenchymal stem cells. to provide.

The present invention provides a food composition for preventing or ameliorating megakaryocyte aplasia comprising a conditioned medium for tonsil-derived mesenchymal stem cells.

The present invention provides a food composition for preventing or improving thrombocytopenia, comprising a conditioned medium for tonsil-derived mesenchymal stem cells.

While the present inventors were contemplating a treatment plan for symptoms related to thrombocytopenia, which is the most problematic symptom among side effects caused by radiation therapy or administration of anticancer drugs, especially high doses of anticancer drugs, the conditioned medium of tonsil-derived mesenchymal stem cells It has a different profile compared to the derived mesenchymal stem cells, thereby greatly improving the side effects of anticancer drugs or radiotherapy, confirming that the effect as an adjuvant for anticancer treatment is remarkable, and completed the present invention.

The present invention is a placental growth factor (PlGF) comprising the step of culturing tonsil-derived mesenchymal stem cells (T-MSC) in a medium containing low glucose DMEM (Low glucose DMEM) ) provides a method for producing a T-MSC conditioned medium with increased expression.

In the present invention, low glucose DMEM (Low glucose DMEM) refers to a DMEM containing glucose (eg, D-glucose) in a small amount compared to high glucose DMEM (High glucose DMEM), generally high concentration glucose DMEM means that it contains 4.5 g/L or more of glucose. The amount of glucose contained in the low-glucose DMEM is not limited, but preferably 0.1 g/L or more and less than 4.5 g/L in DMEM, more preferably 0.5 g/L to 4.0 g/L in DMEM, even more preferably contains 0.5 g/L to 3.0 g/L of glucose in DMEM, most preferably 0.5 g/L to 2.0 g/L of glucose in DMEM.

The present invention also provides a pharmaceutical composition for adjuvant anticancer treatment comprising a conditioned medium for tonsil-derived mesenchymal stem cells.

More specifically, the present invention provides a pharmaceutical composition for adjuvant anticancer treatment to promote platelet production, comprising a conditioned medium for tonsil-derived mesenchymal stem cells with increased expression of placental forming factor (PlGF).

The present invention also provides a method for promoting platelet production comprising administering to a subject in need thereof a conditioned medium for tonsil-derived mesenchymal stem cells.

The present invention also provides a method for promoting megakaryocyte production, comprising administering to a subject in need thereof a conditioned medium for tonsil-derived mesenchymal stem cells.

The present invention also provides a method for preventing or treating megakaryocytic aplasia, comprising administering to a subject in need thereof a conditioned medium for tonsil-derived mesenchymal stem cells.

The present invention also provides a method for preventing or treating thrombocytopenia, comprising administering to a subject in need thereof a conditioned medium for tonsil-derived mesenchymal stem cells.

The present invention also provides for use in therapy of the conditioned medium of tonsil-derived mesenchymal stem cells.

In the present invention, tonsil-derived mesenchymal stem cells are tissues that are located in the back of the nose and the inside of the neck to primarily defend our body from substances such as bacteria that invade from the outside and at the same time act as a lymphepithelial immune tissue. It refers to undifferentiated stem cells with the ability to differentiate into two or more new cells while having the ability to self-replicate derived from the amygdala.

In the present invention, "tonsillage-derived mesenchymal stem cell conditioned medium" means a culture medium obtained by culturing tonsil-derived mesenchymal stem cells, preferably, a culture medium from which tonsil-derived mesenchymal stem cells are removed or a supernatant thereof. it means. It can be used interchangeably with "tonsillium-derived mesenchymal stem cell culture supernatant", "tonsil-derived mesenchymal stem cell conditioned culture medium" or "tonsil-derived mesenchymal stem cell culture medium".

As used herein, the term "anti-cancer therapy adjuvant" or "anti-cancer adjuvant" may refer to use to suppress or improve the side effects of anticancer agents, through which the anticancer effect of anticancer agents may include increasing Accordingly, the composition for adjuvant anticancer treatment may be for reducing, improving, preventing or treating side effects caused by anticancer agents. The anti-cancer adjuvant may be administered in combination with any kind of anti-cancer agent or anti-cancer adjuvant to enhance the anti-cancer effect of the anti-cancer agent and suppress or improve the side effects of the anti-cancer agent.

In the present invention, "cancer treatment aid" means an effect that can promote safer cancer treatment by preventing or treating side effects that may be caused by administration of a pharmaceutical composition for cancer treatment or radiation therapy. In the present invention, anti-cancer treatment adjuvant is to suppress the side effects caused by the administration of the anti-cancer agent or radiation treatment.

For example, it may mean that it can exhibit an effect of preventing or treating immune system side effects caused by the administration of an anticancer agent.

For example, suppression of side effects of anticancer drugs such as hematopoietic toxicity, anemia, thrombocytopenia, and neutropenia caused by the administration of anticancer drugs may be mentioned.

More specifically, the anticancer adjuvant use according to the present invention has an effect of promoting platelet production, and thus has an excellent effect as an adjuvant to anticancer drug administration or radiotherapy.

More specifically, the anticancer adjuvant use according to the present invention has an effect of suppressing thrombocytopenia, and thus has an excellent effect as an adjuvant role for anticancer drug administration or radiotherapy.

More specifically, the anticancer adjuvant use according to the present invention has an effect of increasing megakaryocyte formation, and thus has an excellent effect as an adjuvant to anticancer drug administration or radiotherapy.

More specifically, the anticancer adjuvant use according to the present invention has an effect of inhibiting the reduction of megakaryocytes, and thus has an excellent effect as an adjuvant to anticancer drug administration or radiotherapy.

The present invention also provides a pharmaceutical composition for preventing or treating megakaryocytic aplasia comprising a conditioned medium of tonsil-derived mesenchymal stem cells.

The present invention also provides a pharmaceutical composition for preventing or treating thrombocytopenia comprising a conditioned medium for tonsil-derived mesenchymal stem cells.

As used herein, the term "suppression" refers to any action that reduces the progress of a specific condition or disease (eg, reduces the side effects of anticancer drugs or radiation by administration of an anticancer adjuvant), and in the present invention, the term " "Improvement" means any action in which a specific condition or disease is beneficially changed due to a decrease in the progression of a specific condition or disease (eg, reduction of side effects of anticancer drugs or radiation by administration of anticancer adjuvants or reduction of side effects of anticancer drugs or radiation to cancer due to reduction of side effects) Any action that improves or changes to a beneficial effect).

As used herein, "treat" refers to an individual suffering from or at risk of developing a disease, ameliorating the individual's condition (eg, one or more symptoms), delaying disease progression, symptoms means any form of treatment or prophylaxis that provides an effect including, but not limited to, delaying the onset or slowing the progression of symptoms. Accordingly, the term “treatment” also includes prophylactic treatment of an individual that prevents the occurrence of symptoms.

As used herein, “treatment” and “prevention” are not intended to mean cure or complete elimination of a symptom. They refer to any form of treatment that provides an effect to a patient suffering from a disease, including amelioration of the patient's condition (eg, one or more symptoms), delaying the progression of the disease, and the like.

As used herein, "treatment-effective amount" refers to an amelioration of a side effect or condition (eg, one or more symptoms) in a subject or patient suffering from or at risk of developing a disease, delaying the progression of the disease, etc. means an amount sufficient to produce the desired effect, including

Cancer is a mass or tumor composed of undifferentiated cells that proliferate indefinitely within a tissue. It can be used for the treatment of colon cancer, liver cancer, thyroid cancer, gallbladder cancer, biliary tract cancer, pancreatic cancer, prostate cancer, esophageal cancer, cervical cancer, colon cancer, bladder cancer, central nerve tumor, or brain tumor.

The anti-cancer therapeutic adjuvant according to the present invention may include administration of an anti-cancer agent, for example, administration of a high-dose anti-cancer agent or administration of a long-term anti-cancer agent; Alternatively, it may be to provide an conditioned medium for tonsil-derived mesenchymal stem cells as an adjuvant for improving side effects caused by radiation therapy.

Specifically, the bone marrow corresponds to an organ in which cell production is active and is greatly affected by anticancer drugs or radiation therapy. Accordingly, the production of platelets produced in the bone marrow may be hindered by anticancer drugs. Accordingly, there is a high possibility that symptoms and risks caused by a lack of platelets will occur. That is, the anticancer treatment adjuvant according to the present invention may be inhibition of platelet reduction by administration of an anticancer agent or radiation treatment. More specifically, it may be inhibition of platelet reduction by high-dose anticancer drug administration or radiation therapy.

According to an embodiment of the present invention, it was confirmed that the conditioned medium of the present invention significantly increased only the production of platelets, unlike the case of red blood cells and white blood cells.

Platelets are non-nucleated cells present in mammalian blood as fragments of cells that are produced by megakaryocytes and are involved in blood coagulation and inflammatory reactions. Platelets mediate blood clot formation and hemostasis, play an important role in repair and regeneration of connective tissue, and release growth factors that promote wound healing. A decrease in platelets means a decrease in platelets as blood cells that act as hemostats when bleeding occurs, and can cause problems such as easy bruising, nosebleeds, and bleeding gums.

The anticancer agent is busulfan, abiraterone acetate, afatinib, aldesleukin, alemtuzumab, alitretinoin, altretamine, ami Amifostine, Aminoglutethimide Anagrelide, Anastrozole, Arsenic Trioxide, Asparaginase, Azacitidine, Azathioprine, bendamustine, bevacizumab, bexarotine, bicalutamide, bleomycin, bortezomib, busulfan (Busulfan), Capecitabine, Carboplatin, Carmustine, Cetuximab, Chlorambucil, Cisplatin, Cladribine , Crizotinib, Cyclophosphamide, Cytarabine, Dacarbazine, Dactinomycin, Dasatinib, Daunorubicin, Denileukin diftitox, Decitabine, Docetaxel, Dexamethasone, Doxifluridine, Doxorubicin, Epirubicin, Epirubicin Epoetin Alpha, Epothilone, Erlotinib, Estramustine, Etinost at), Etoposide, Everolimus, Exemestane, Filgrastim, Floxuridine, Fludarabine, Fluorouracil ), Fluoxymesterone, Flutamide, folate-bound alkaloids, Gefitinib, Gemcitabine, Gemtuzumab ozogamicin, GM-CT- 01, Goserelin, Hexamethylmelamine, Hydroxyureas, Ibritumomab, Idarubicin, Ifosfamide, Imatinib , interferon alpha, interferon beta, irinotecan, ixabepilone, lapatinib, leucovorin, leuprolide, lenalidomide, letrozole ( Letrozole), Lomustine, Mechlorethamine, Megestrol, Melphalan, Mercaptopurine, Methotrexate, Mitomycin, Mitoxane Mitoxantrone, Nelarabine, Nilotinib, Nilutamide, Octreotide, Ofatumumab, Oprelvekin, Oxaliplatin , Paclitaxel, Panitumumab, Pemetrexed, Pentostatin, Polysaccharide galectin inhibitor, Procarbaz ine), Raloxifene, Retinoic acids, Rituximab, Romiplostim, Sargramostim, Sorafenib, Streptozocin, Sunitinib, Tamoxifen, Temsirolimus, Temozolamide, Teniposide, Thalidomide, Thioguanine, Thiotepa ), thioguanine, topotecan, toremifene, tositumomab, trametinib, trastuzumab, tretinoin, valrubicin ), VEGF inhibitors and traps, vinblastine, vincristine, vindesine, vinorelbine, vintafolde (EC145), vorinostat (Vorinostat) ), a salt thereof, or any combination thereof.

More specifically, busulfan; Cyclophosphamide; anticancer agents containing platinum atoms such as carboplatin, cisplatin, and oxaliplatin; And it may be any one or more anticancer agents selected from the group consisting of angiogenesis-inhibiting monoclonal antibody-based bevacizumab.

That is, it may be any one or more anticancer agents selected from the group consisting of busulfan, cyclophosphamide, carboplatin, cisplatin, oxaliplatin, and bevacizumab.

The pharmaceutical composition for adjuvant anticancer treatment according to the present invention promotes the differentiation (Megakaryopoiesis) of bone marrow cells damaged by anticancer agents or radiation into megakaryocytes and greatly increases the number of platelets, thereby having an excellent role as a treatment adjuvant.

In particular, the conditioned medium for tonsil-derived mesenchymal stem cells according to the present invention has a different protein profile compared to other-derived mesenchymal stem cells and their conditioned medium.

Specifically, the conditioned medium for tonsil-derived mesenchymal stem cells contains a large amount of placental growth factor (PlGF), unlike other derived mesenchymal stem cells and their medium. PlGF is a component of the vascular endothelial growth factor (VEGF) family and is involved in angiogenesis. It is also involved in the maturation of megakaryocytes and promotion of platelet production. Accordingly, the tonsil stem cell culture medium containing PlGF has an excellent action as an adjuvant for anticancer treatment that prevents bleeding by promoting platelet production.

In addition, the tonsil-derived mesenchymal stem cells according to the present invention are easier to obtain than the other-derived stem cells, which is the tonsil, the tonsil tissue discarded after surgery can be recycled, and the initial yield is very high. The amount of medium can be freely adjusted.

In the present invention, the conditioned medium for tonsil-derived mesenchymal stem cells includes those prepared through isolation, culture, and special manipulation from the subject, and is used as a drug used for the purpose of treatment, diagnosis and prevention to be used as an adjuvant for anticancer treatment. can

In the present invention, tonsil-derived mesenchymal stem cells can be extracted from tonsils according to methods known in the art. For example, it can be extracted from the tonsils according to the method described in Korean Patent No. 10-1508413 and the like.

The conditioned medium for tonsil-derived mesenchymal stem cells may be a culture solution obtained after culturing tonsil-derived mesenchymal stem cells and removing the cells, a culture supernatant or a concentrate thereof, or a lyophilisate thereof.

Tonsil-derived mesenchymal stem cells can be conventionally cultured using a culture medium for stem cells. The tonsil-derived mesenchymal stem cell culture solution may be obtained by culturing tonsil-derived mesenchymal stem cells obtained from tonsil tissue in serum or iron-free medium. The supernatant obtained after removing the stem cells and macromolecules by centrifugation or filtration using a filter can be used. In addition, the obtained supernatant can be used as it is or used as a concentrate obtained by concentration.

Culture medium and culture conditions for culturing tonsil-derived mesenchymal stem cells are well known in the art to which the present invention pertains, and those of ordinary skill in the art may appropriately select or modify them.

The medium is a medium suitable for maintaining and storing the same cell type as the tonsil-derived mesenchymal stem cells, and may be a DMEM medium supplemented with serum. The serum may be fetal bovine serum (FBS), but is not limited thereto, and may be 1 to 10% by weight based on the total weight of the serum medium. If necessary, antibiotics, antifungals and mycoplasma inhibitors may be included, and these may be 1% by weight based on the total weight of the medium. Antibiotics include antibiotics commonly used in cell culture, such as penicillin-streptomycin, antifungal agents include amphotericin-B (fungizone), etc., and mycoplasma inhibitors include gentamicin, ciprofloxacin, tylosin, etc. , but is not limited thereto.

The medium may be a serum-free medium. Preferably, the serum-free medium may be a low glucose DMEM medium.

According to one embodiment of the present invention, the tonsil-derived mesenchymal stem cell conditioned medium is a concentrated supernatant from which cells are removed by culturing tonsil-derived mesenchymal stem cells in serum-free low glucose DMEM.

The pharmaceutical composition of the present invention may be formulated in various formulations such as solutions, suspensions, emulsions, lotions, ointments, and freeze-drying agents according to conventional methods.

The pharmaceutical composition of the present invention may be formulated and administered in a unit dosage form suitable for administration in the body of a patient according to a conventional method in the pharmaceutical field, and the preparation may be administered effectively by one or several administrations. includes the amount As a formulation suitable for this purpose, an injection, an injection, etc. are preferable as a formulation for parenteral administration. In addition, the pharmaceutical composition for adjuvant anticancer treatment may include a pharmaceutically acceptable conventional inert carrier and diluent. Pharmaceutically acceptable carriers and diluents that may be included in the pharmaceutical composition of the present invention include excipients such as starch, sugar, and mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin, etc. , alginate, and binders such as polyvinyl pyrrolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, interfacial agents such as polysorbate, cetyl alcohol, and glycerol active agents, but are not limited thereto. The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the recipient to which it will be transplanted. Diluents include, but are not limited to, saline, aqueous buffers, solvents, and/or dispersion media. In addition, for example, in the case of injections, preservatives, analgesics, solubilizers or stabilizers, etc., and in the case of formulations for topical administration, a base, excipients, lubricants or preservatives may be further included. The compositions of the present invention may be used undisturbed or frozen for later use. If frozen, standard cryopreservatives (eg DMSO, glycerol, Epilife® Cell Freezing Medium (Cascade Biologics)) can be added to the cell population prior to freezing.

In addition, it may be administered using an administration method commonly used in the art, and preferably, direct administration to the diseased site of the subject in need of treatment is possible, but is not limited thereto. In addition, the administration can be both non-surgical administration using a catheter and surgical administration methods such as injection after incision at the diseased site. The dosage may vary depending on the degree of concentration, but 10 μl/kg to 1 ml/kg of body weight may be administered once or divided into several doses.

However, it should be understood that the actual dosage of the active ingredient should be determined in light of several related factors such as the disease to be treated, the severity of the disease, the route of administration, the subject's weight, age, and sex, and therefore, the dosage may be any It is not intended to limit the scope of the present invention in any way.

The present invention also provides a food composition for adjuvant anticancer treatment comprising a conditioned medium for tonsil-derived mesenchymal stem cells. More specifically, more specifically, the present invention provides a food composition for adjuvant anti-cancer treatment for promoting platelet production, comprising a conditioned medium for tonsil-derived mesenchymal stem cells.

The present invention also provides a food composition for preventing or improving megakaryocytic aplasia, comprising a conditioned medium for tonsil-derived mesenchymal stem cells.

The present invention also provides a food composition for preventing or improving thrombocytopenia, comprising a conditioned medium for tonsil-derived mesenchymal stem cells.

The present invention can be generally used as a commonly used food product.

The food composition of the present invention can be used as a health functional food. The term "health functional food" means a food manufactured and processed using raw materials or ingredients useful for the human body in accordance with the Health Functional Food Act, and "functionality" refers to the structure and function of the human body. It refers to ingestion for the purpose of obtaining useful effects for health purposes such as regulating nutrients or physiological effects.

The food composition of the present invention may include conventional food additives, and the suitability as the "food additive" is determined according to the general rules and general test methods of food additives approved by the Ministry of Food and Drug Safety, unless otherwise specified. It is judged according to the standards and standards related to the item.

The items listed in the “Food Additives Code” include, for example, chemical compounds such as ketones, glycine, potassium citrate, nicotinic acid, and cinnamic acid; Mixed preparations such as sodium L-glutamate preparation, noodle-added alkali agent, preservative agent, and tar color agent can be mentioned.

The food composition of the present invention may contain 0.01 to 95% by weight of the conditioned medium for tonsil-derived mesenchymal stem cells, preferably 5 to 90% by weight, based on the total weight of the composition.

In addition, the food composition of the present invention can be manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of adjuvant anticancer treatment.

For example, among health functional foods in the form of capsules, hard capsules can be prepared by filling a conventional hard capsule with a mixture with an additive such as a conditioned medium for tonsil-derived mesenchymal stem cells according to the present invention and an excipient, Soft capsules can be prepared by filling a mixture with additives such as the food composition and excipients according to the present invention in a capsule base such as gelatin. The soft capsule formulation may contain a plasticizer such as glycerin or sorbitol, a colorant, a preservative, and the like, if necessary.

The term definitions for the excipients, binders, disintegrants, lubricants, flavoring agents, flavoring agents, etc. are described in documents known in the art and include those having the same or similar functions (Explanation of the Korean Pharmacopoeia, Moonseongsa, Korea) Association of Colleges of Pharmacy, 5th ed., p33-48, 1989).

There is no particular limitation on the type of food, and includes all health functional foods in the ordinary sense.

The present invention provides an adjuvant method for anticancer treatment comprising administering to a subject a conditioned medium for tonsil-derived mesenchymal stem cells.

In the present invention, terms such as "conditioned medium for tonsil-derived mesenchymal stem cells", "anticancer treatment adjuvant", and "administration" are the same as described above.

The subject refers to an animal, and typically may be a mammal that can exhibit beneficial effects by treatment using the conditioned medium for tonsil-derived mesenchymal stem cells of the present invention. Preferred examples of such subjects may include primates such as humans. In addition, such subjects may include all subjects with or at risk of having symptoms of cancer.

The present invention also provides the use of a conditioned medium for tonsil-derived mesenchymal stem cells in the manufacture of a medicament for adjuvant anticancer therapy.

The present invention also provides a composition comprising a conditioned medium for tonsil-derived mesenchymal stem cells for use in adjuvant anticancer therapy.

The present invention also provides the use of an conditioned medium for tonsil-derived mesenchymal stem cells for adjuvant anticancer therapy.

The composition of the present invention has a marker and/or protein profile different from that of other derived mesenchymal stem cells or their conditioned media, and exhibits an action effect such as promoting megakaryocyte formation, inhibiting and enhancing thrombocytopenia, and has an excellent effect in adjuvant anticancer treatment have

1 shows the results of confirming the expression level of PlGF from tonsil-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells and bone marrow-derived mesenchymal stem cells, and their conditioned medium.

Figure 2 is the result of confirming the expression level and CD marker expression pattern of the growth factor in the stem cell conditioned medium of various origins. Figure 2a shows the result of confirming the expression level of PlGF from the conditioned medium of tonsil-derived mesenchymal stem cells, adipose-derived mesenchymal stem cells, bone marrow-derived mesenchymal stem cells, and Wharton's jelly cells. Figure 2b is the result of comparing the CD marker expression of T-MSC, AD-MSC, BM-MSC, UC-MSC-derived mesenchymal stem cells (MSCs) in a heat map (heat map).

3 shows the results of confirming the changes in red blood cells, white blood cells, and platelets according to the treatment of tonsil-derived mesenchymal stem cells after administration of a high-dose anticancer agent.

4 shows the results of confirming the change in the number of CD41+ megakaryocytes in the bone marrow according to the treatment of tonsil-derived mesenchymal stem cells after administration of a high-dose anticancer agent.

5 is Shows the results of confirming the effect of promoting megakaryocyte formation by PlGF in the conditioned medium of tonsil-derived mesenchymal stem cells.

Examples and preparation examples are presented to help the understanding of the invention. The following Examples and Preparation Examples are only provided for easier understanding of the present invention, and the content of the present invention is not limited by the Examples and Preparation Examples.

<Example 1> Preparation of adipose, bone marrow or tonsil-derived mesenchymal stem cells or their conditioned medium

Tonsil-derived mesenchymal stem cells (T-MSC) are derived from tonsil tissue (low-age tissue aged 4-20 years old) extracted from a patient undergoing tonsillectomy at the Department of Otolaryngology, Throat, Head and Neck, Ewha Womans University Mokdong Hospital. got it This was manufactured similarly to the manufacturing method of Korean Patent No. 10-1508413.

To prepare an conditioned medium from the stem cells prepared above, tonsil-derived mesenchymal stem cells were cultured with low glucose DMEM (10% FBS, 100 IU/ml penicillin and 100 ug/ml streptomycin). Low glucose DMEM used was one containing glucose at a concentration of 1,000 mg/L, and was purchased from Wellgene (Gyeongsan, Gyeongbuk, Korea). When the 100 mm culture tissue plate reached 70-80% confluency, the medium was removed, washed four times with PBS while the cells were attached to the plate, and then further cultured with low glucose DMEM for 48 hours. Then, the medium is rolled up, centrifuged at 5000 rpm for 100 minutes, and the supernatant is filtered with a 0.2 uM filter. The filtered medium was concentrated 20 times by centrifugal filtration (cut-off of 3K, Amicon Ultra-15, Millipore) for use in the experiment or stored at -80°C.

For adipose-derived mesenchymal stem cells (AT-MSC), cells of the same passage provided by RNL Bio were used, and in the case of bone marrow mesenchymal stem cells, cells of the same passage provided by the Severance Hospital Cell Therapy Center, Yonsei University, Seoul were used. , Wharton's jelly (PromoCell, Heidelgerg, Germany) was also used in the experiment. For the preparation of the conditioned medium, it was prepared under the same conditions as the conditioned medium from gastric tonsil-derived mesenchymal stem cells.

<Example 2> Confirmation of characteristics of conditioned medium and CD markers of tonsil-derived mesenchymal stem cells

In order to confirm the characteristics of the conditioned medium of stem cells of different origins, the difference in secreted proteins was confirmed through Western blot and growth factor array.

Specifically, the conditioned medium and each cell obtained from bone marrow mesenchymal stem cells, adipose mesenchymal stem cells, and tonsil stem cells were electrophoresed on 10% SDS PAGE to separate proteins and transferred to PVDF membrane (Millipore). The transferred membrane was blocked in 5% skim milk powder for 1 hour and then washed 3 times with TBST solution. After reacting with the primary antibody, PlGF (1:250, sc-518003; Santa Cruz Biotechnology, Santa Cruz, CA, USA) at 4°C overnight, washed 3 times with TBST solution and the secondary antibody, anti-mouse antibody (HRP) -conjugated) and reacted at room temperature for 1 hour. After washing three times with TBST, the colors were developed with EZ-Western Lumi Femto Kit (DoGen Bio, Seoul, Korea), and images were taken with ImageQuant LAS 3000 (Fujifilm).

For growth factor arrays, cells were incubated with serum-free DMEM for 48 hours and concentrated using a centrifugal filter device. The protein concentration of the conditioned medium was quantified using a Pierce BCA protein assay kit (ThermoFisher Scientific, Waltham, MA, USA) and normalized to 3 mL of the conditioned medium secreted from 1×10 ⁶ MSCs. RayBiotech label-based human growth factor arrays (Peachtree Corners, GA, USA) were performed according to the manufacturer's instructions. Glass slides were scanned using a GenePix 4000B Microarray Scanner (Molecular Devices, San Jose, CA) and analyzed using a GenePix Pro 7 (Molecular Devices). In the above analysis, Wharton's jelly (PromoCell, Heidelgerg, Germany) was also analyzed.

The results are shown in FIGS. 1 and 2 .

1 shows the results confirmed by Western blot. As can be seen in FIG. 1 , it was confirmed that the expression level of PlGF was high in both tonsil-derived mesenchymal stem cells and their CMs (TM1, TM2), that is, in both Cellular and Secreted forms. On the other hand, no such expression was observed in adipose-derived (AM) or bone marrow-derived (BM) cells.

In addition, as can be seen in FIG. 2a , as a result of analysis through the growth factor array, the expression level of PlGF was high only in the conditioned medium of tonsil-derived mesenchymal stem cells, and adipose-derived (AM), bone marrow-derived (BM) Or it was confirmed that almost no expression in the conditioned medium derived from Wharton's jelly (WJ).

Additionally, the expression of CD markers in target cells was confirmed through flow analysis (Flow Cytometry or FACS; Fluorescence-activated cell sorting) using T-MSC, AD-MSC, BM-MSC, and UC-MSC-derived mesenchymal stem cells. did. As can be seen in FIG. 2b , it was confirmed that the expression patterns of CD markers between T-MSC and AD-MSC, BM-MSC, and UC-MSC-derived mesenchymal stem cells were significantly different.

<Example 3> Confirmation of side effects improvement by administration of high-dose anticancer drugs by administration of conditioned medium for tonsil-derived mesenchymal stem cells

8-week-old female BALB/c mice were purchased from Orient Bio (Seongnam, Republic of Korea) and received food and water ad libitum at a temperature of 21-23°C, a humidity of 51-54%, and a light/dark cycle of 12 hours under normal conditions. was bred with

The mice were intraperitoneally injected with the anticancer drug cyclophosphamide (80 mg/kg/day) for 3 days. Some mice in the cyclophosphamide administration group were intraperitoneally injected with the conditioned medium (CM) of <Example 1> on the 1st and 4th days. Mice were sacrificed on day 7 of the start of the experiment. Experiments and procedures were approved by the Animal Ethics Committee (EUM20-014), Ewha Womans University College of Medicine, and all experiments were performed in accordance with the relevant guidelines and regulations. injected.

The time of administration of the conditioned medium for tonsil-derived mesenchymal stem cells was set as day 0, and the number of red blood cells, white blood cells and platelets after the start of the experiment was measured with Auto Hematology Analyzer (BC-2800Vet; Mindray, Shenzhen, China). In addition, bone marrow cells were isolated from mouse femurs and stained with FITC-conjugated anti-mouse CD41 antibody (Biolegend, San Diego, CA, USA), and the level of CD41+ megakaryocytes in the bone marrow was confirmed through flow cytometry.

(1) Check red blood cell and white blood cell changes

Seven days after the start of the experiment, the number of blood cells was checked in the peripheral blood.

Specifically, it was confirmed that red blood cells and white blood cells were decreased by the administration of anticancer drugs. However, it was confirmed that the platelet count was significantly increased in the group to which the conditioned medium of tonsil-derived mesenchymal stem cells was administered.

The results are shown in FIG. 3 .

As can be seen in FIG. 3 , when the conditioned medium for tonsil-derived mesenchymal stem cells according to the present invention was administered, the number of platelets significantly decreased by the administration of the anticancer agent was greatly increased.

(2) increase the number of CD41+ megakaryocytes in the bone marrow

The level of CD41+ megakaryocytes in the bone marrow was confirmed through flow cytometry, and the results are shown in FIG. 4 .

As can be seen in FIG. 4 , it was confirmed that the number of CD41+ megakaryocytes significantly increased due to administration of the conditioned medium for tonsil-derived mesenchymal stem cells according to the present invention. It was confirmed that the conditioned medium of tonsil-derived mesenchymal stem cells had an excellent improvement effect on the reduction of megakaryocyte formation caused by high-dose anticancer drugs.

<Example 4> Confirmation of effect of promoting formation of megakaryocytes by PlGF in conditioned medium of tonsil-derived mesenchymal stem cells

Bone marrow cells were obtained from the femur and long bones of C57BL/6 mice and cultured in RPMI 1640 media (10% FBS, 2 mM L-glutamine, 10 μM HEPES). In order to induce bone marrow cell damage by anticancer drugs, busulfan was treated at a concentration of 30 μM for 6 hours and cultured, followed by MegaCult-C assay (STEMCELL Technologies). To confirm the effect of PlGF in the tonsil stem cell culture medium and culture medium on megakaryocyte formation, 1 μg/mL concentration of Normal Goat IgG or human PlGF antibody (AF-264, R&D systems) was added to the tonsil stem cell culture medium at 37 °C. After culturing for 7 days in a CO ₂ incubator, megakaryocyte staining was performed.

After the mouse bone marrow cells were separated and treated with busulfan, the change in megakaryocyte-forming ability by the tonsil stem cell culture was confirmed and shown in FIG. 5 . It was confirmed that the megakaryocyte-forming ability of bone marrow cells was decreased by pretreatment with busulfan, and megakaryocyte-forming ability increased to a level similar to that of the control group when megakaryocyte differentiation was induced with amygdala stem cell culture medium. When the PlGF neutralizing antibody was treated together with the tonsil stem cell culture medium, it was confirmed that the effect of enhancing the megakaryocyte formation ability by the tonsil stem cell culture medium was decreased. As confirmed above, the conditioned medium for tonsil-derived mesenchymal stem cells has an excellent therapeutic effect on the reduction of megakaryocyte formation caused by high-dose anticancer drug treatment.

In particular, the effect of the conditioned medium for tonsil-derived mesenchymal stem cells was confirmed to support the above-mentioned effects by containing a large amount of PlGF, etc., a factor not found in the conditioned media of bone marrow-derived or adipose-derived mesenchymal stem cells.

Claims (23)

Tonsil-derived mesenchymal stem cells (T-MSC) expression of placental growth factor (PlGF) comprising the step of culturing in a medium containing low glucose DMEM (Low glucose DMEM) Method for preparing this increased T-MSC conditioned medium. The method according to claim 1, wherein the concentration of glucose is 0.1 g/L or more and less than 4.5 g/L in DMEM. The method according to claim 2, wherein the concentration of glucose is 0.5 g/L to 4.0 g/L in DMEM. A pharmaceutical composition for adjuvant anticancer treatment comprising the conditioned medium prepared by the method of claim 1. [Claim 5] The pharmaceutical composition for adjuvant anticancer treatment according to claim 4, wherein the adjuvant to anticancer treatment is characterized by promoting platelet production. The pharmaceutical composition for adjuvant anticancer treatment according to claim 4, wherein the anticancer treatment is radiation treatment or administration of an anticancer agent. According to claim 6, wherein the anticancer agent is busulfan, cyclophosphamide (Cyclophosphamide), carboplatin (Carboplatin), cisplatin (Cisplatin), oxaliplatin (Oxaliplatin) and any one or more selected from the group consisting of bevacizumab Characterized in, a pharmaceutical composition for adjuvant anticancer treatment. The pharmaceutical composition for adjuvant anticancer treatment according to claim 4, wherein the adjuvant to anticancer treatment is inhibition of platelet reduction by administration of an anticancer agent or radiation treatment. [Claim 5] The pharmaceutical composition for adjuvant anti-cancer treatment according to claim 4, wherein the anti-cancer treatment adjuvant is characterized in that the reduction of megakaryocytes by anti-cancer drug administration or radiation treatment is suppressed. A pharmaceutical composition for the prevention or treatment of megakaryocytic aplasia comprising the conditioned medium prepared by the method of claim 1 . A pharmaceutical composition for the prevention or treatment of thrombocytopenia comprising the conditioned medium prepared by the method of claim 1 . A food composition for adjuvant anticancer treatment comprising the conditioned medium prepared by the method of claim 1. [Claim 13] The food composition for adjuvant anti-cancer treatment according to claim 12, wherein the anti-cancer treatment aid is characterized by promoting platelet production. The food composition for adjuvant anticancer treatment according to claim 12, wherein the anticancer treatment adjuvant is characterized by increased megakaryocyte formation. A food composition for preventing or improving megakaryocytic aplasia comprising the conditioned medium prepared by the method of claim 1 . A food composition for preventing or improving thrombocytopenia, comprising the conditioned medium prepared by the method of claim 1 . A method of adjuvant anticancer treatment comprising administering to a subject in need thereof the conditioned medium prepared by the method of claim 1 . A method for promoting platelet production, comprising administering the conditioned medium prepared by the method of claim 1 to a subject in need thereof. A method for promoting megakaryocyte production comprising administering the conditioned medium prepared by the method of claim 1 to a subject in need thereof. A method for preventing or treating megakaryocytic aplasia, comprising administering the conditioned medium prepared by the method of claim 1 to a subject in need thereof. A method for preventing or treating thrombocytopenia, comprising administering the conditioned medium prepared by the method of claim 1 to a subject in need thereof. The therapeutic use of the conditioned medium prepared by the method of claim 1. Use of a conditioned medium for tonsil-derived mesenchymal stem cells in the manufacture of a medicament for adjuvant cancer treatment.

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