Agent for improving cancer cachexia and method for improving cancer cachexia

Abstract

A novel agent for improving cancer cachexia, which is an agent for improving cancer cachexia for administration to a subject suffering from cancer cachexia, the agent for improving cancer cachexia being a composition comprising culture supernatant of stem cells derived from dental pulp, wherein the culture supernatant comprises microparticles derived from the above-mentioned stem cells derived from dental pulp; and a method for improving cancer cachexia.

Description

Technical Field

[0001] The present invention relates to an agent for improving cancer cachexia (cachexia) and a method for improving cancer cachexia. Background Art

[0002] Cancer cachexia, often referred to as cancer anorexia-cachexia syndrome (CACS), is a multifactorial symptom of advanced cancer. Symptoms of cancer cachexia include weight loss (weight loss), in particular, poor nutritional status, anemia, and the like.

[0003] As a method for treating cachexia in late-stage cancer, it is required to improve or alleviate these symptoms to improve QOL (Quality of Life) or ADL (Activities of Daily Living) during survival as much as possible, even if the survival period is not prolonged.

[0004] As a method for treating cachexia, Patent Document 1 describes a method for treating cachexia in a patient, the method comprising administering a therapeutically effective amount of anamorelin to a human cancer patient once a day during an effective treatment period.

[0005] Prior art literature

[0006] Patent Literature

[0007] Patent Document 1: Japanese Patent Application No. 2019-535691;

[0008] Patent Document 2: Japanese Patent Application No. 2019-514416;

[0009] Patent document 3: Japanese Patent Application Publication No. 2018-154655. Summary of the invention

[0010] Problems to be solved by the invention

[0011] On the other hand, methods for treating cachexia using substances other than anamorelin have been studied. In addition, Patent Document 1 also describes the use of small molecule compounds such as megestrol acetate, melatonin, and ostarine for treating cachexia. In addition, Patent Documents 2 and 3 disclose pharmaceutical compositions containing extracellular vesicles secreted by mesenchymal stem cells, and cachexia is exemplified as an applicable disease (claim 6 of Patent Document 2,

[0210] of Patent Document 3, etc.).

[0012] The problem to be solved by the present invention is to provide a novel agent for improving cancer cachexia.

[0013] Means for solving problems

[0014] The present inventors have found that the culture supernatant of dental pulp-derived stem cells or microparticles such as exosomes derived from the culture supernatant can improve various symptoms of cachexia in cancer cachexia model mice.

[0015] It should be noted that the effects of dental pulp-derived stem cells on cachexia are not described in Patent Documents 2 and 3. In the examples of Patent Document 2, it is described in

[0227] that DP-MSC (exosomes derived from dental pulp stem cells) have a low effect, but the performance is the same as that of UC- (stem cells derived from the umbilical cord) and CH-MSC (exosomes derived from placental ciliary membrane stem cells), and there is no example of using dental pulp-derived stem cells to study cachexia. In Patent Document 3, there is no example of using exosomes obtained from the culture supernatant of dental pulp-derived stem cells.

[0016] Specifically, the present invention and preferred configurations of the present invention are as follows.

[0017] [1] An agent for improving cancer cachexia, the agent for improving cancer cachexia being for administration to a subject suffering from cancer cachexia,

[0018] The cancer cachexia improving agent is a composition comprising a culture supernatant of stem cells derived from dental pulp,

[0019] The culture supernatant contains microparticles derived from dental pulp-derived stem cells.

[0020] [2] An agent for improving cancer cachexia, the agent for improving cancer cachexia being for administration to a subject suffering from cancer cachexia,

[0021] The cancer cachexia improving agent is a composition containing microparticles derived from the culture supernatant of dental pulp-derived stem cells.

[0022] The composition does not contain a substance obtained by removing fine particles from the culture supernatant.

[0023] [3] The agent for improving cancer cachexia according to [1] or [2], wherein the microparticles are exosomes.

[0024] [4] The agent for improving cancer cachexia according to any one of [1] to [3], which is used for treating weight loss in a subject suffering from cancer cachexia.

[0025] [5] The agent for ameliorating cancer cachexia according to [4], wherein the subject has lost 2% or more of body weight in the past 14 days.

[0026] [6] The agent for improving cancer cachexia according to any one of [1] to [5], wherein the agent is used for treating anemia in a subject suffering from cancer cachexia.

[0027] [7] The agent for improving cancer cachexia described in [6] is used for suppressing the decrease in red blood cell count, hemoglobin value and hematocrit value in a subject suffering from cancer cachexia.

[0028] [8] The agent for improving cancer cachexia according to any one of [1] to [7], which is used for suppressing increases in IL-6 concentration and TFN-α concentration in the blood of a subject suffering from cancer cachexia.

[0029] [9] The agent for improving cancer cachexia according to any one of [1] to [8], wherein the agent is used for administering the agent for improving cancer cachexia to a subject suffering from cancer cachexia once or more per week during an effective treatment period.

[0030]

[10] A method for improving cancer cachexia, comprising administering an effective amount of the agent for improving cancer cachexia according to any one of [1] to [9] to a subject suffering from cancer cachexia.

[0031]

[11] The method for improving cancer cachexia described in

[10] , wherein the subject suffering from cancer cachexia is an animal other than a human.

[0032] Effects of the Invention

[0033] According to the present invention, a novel agent for improving cancer cachexia can be provided. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] [ Figure 1 ] Figure 1 This is a schematic diagram of an experimental protocol for administering a sample to a cancer cachexia model mouse to evaluate the improvement of cancer cachexia.

[0035] [ Figure 2 ] Figure 2 This is a graph showing the relationship between the number of days from the treatment date and the body weight (% relative to normal mice used as a control) when the cancer cachexia model mice were administered with the cancer cachexia improving agents of Examples 1 and 2 or the sample (physiological saline) of the comparative example.

[0036] [ Figure 3 ] Figure 3 The graph shows the IL-6 concentration in the blood in normal mice and in the case where the cancer cachexia improving agents of Examples 1 and 2 or the sample (physiological saline) of the comparative example were administered to cancer cachexia model mice.

[0037] [ Figure 4 ] Figure 4The graph shows the TFN-α concentration in the blood in normal mice and in cancer cachexia model mice to which the cancer cachexia ameliorating agents of Examples 1 and 2 or the sample of Comparative Example (physiological saline) were administered.

[0038] [ Figure 5 ] Figure 5 The graph shows the relationship between the number of days from the treatment date and the survival rate in the case of normal mice and in the case of cancer cachexia model mice administered with the cancer cachexia improving agents of Examples 1 and 2 or the sample (physiological saline) of the comparative example.

[0039] [ Figure 6 ] Figure 6 The graph shows the relationship between the number of days from the treatment date and the survival rate in the case of normal mice and in the case of cancer cachexia model mice when the dosage of the cancer cachexia ameliorating agent of Example 2 was increased to 1.25 times or when the sample of the comparative example (physiological saline) was administered.

[0040] [ Figure 7 ] Figure 7 The graph shows the concentration of IL-6 released into the culture medium after culturing the activated (activated) macrophages that are the main cause of cytokine storm caused by LPS stimulation, in the case of no treatment (Comparative Example A) and in the case of administration of the cancer cachexia improving agent of Examples 1 and 2 or the samples of the comparative examples (physiological saline of Comparative Example B, culture supernatant or exosomes of umbilical cord stem cells of Comparative Examples C and D). DETAILED DESCRIPTION

[0041] Hereinafter, the present invention will be described in detail. The description of the constituent elements recorded below is sometimes described based on representative embodiments or specific examples, but the present invention is not limited to such embodiments. It should be noted that the numerical range represented by "to" in this specification refers to a range including the numerical values ​​recorded before and after "to" as the lower limit and upper limit.

[0042] [Cancer cachexia improver]

[0043] A first embodiment of the cancer cachexia ameliorating agent of the present invention is an ameliorating agent for use in administering to a subject suffering from cancer cachexia, and is a composition comprising a culture supernatant of dental pulp-derived stem cells, wherein the culture supernatant contains microparticles derived from the dental pulp-derived stem cells.

[0044] The second embodiment of the cancer cachexia ameliorating agent of the present invention is an agent for ameliorating cancer cachexia for administration to a subject suffering from cancer cachexia, which is a composition comprising microparticles derived from the culture supernatant of stem cells from dental pulp, and the composition does not contain a substance obtained by removing the microparticles from the culture supernatant.

[0045] From the viewpoint of enhancing the effect of improving cancer cachexia, the cancer cachexia improving agent of the present invention is more preferably a composition comprising a culture supernatant of dental pulp-derived stem cells, and particularly preferably a culture supernatant of dental pulp-derived stem cells.

[0046] Hereinafter, preferred embodiments of the cancer cachexia ameliorating agent of the present invention will be described.

[0047] <Definition of cancer cachexia>

[0048] Cancer cachexia refers to cachexia in a subject with cancer. It should be noted that the cancer cachexia-improving agent of the present invention is an agent for improving cancer cachexia, but can also improve cachexia other than cancer cachexia.

[0049] "Cachexia" can be defined in various ways in any of the main embodiments or subordinate embodiments of the present invention. In particular, any of the following definitions can be used:

[0050] (1) A clinical syndrome characterized by one or a combination of loss of appetite, early satiety, weight loss, muscle fatigue, malnutrition, anemia and edema, but preferably a clinical syndrome defined by three, four, five or all of these symptoms.

[0051] (2) Weight loss of more than 5% and / or less than 20 kg / m2 in the past 6 months 2 body mass index.

[0052] (3) BMI < 20 and more than 2% weight loss in the past 3 or 6 months.

[0053] (4) In cases consistent with sarcopenia (male <7.26 kg / m 2 ; Female<5.45kg / m 2 ) and have lost >2% of their body weight in the past 3 or 6 months.

[0054] (5) A multifactorial syndrome characterized by severe weight loss, fat and muscle loss, and increased protein degradation (catabolism) caused by the underlying disease.

[0055] Regarding weight loss, when the agent for ameliorating cancer cachexia of the present invention is used to treat weight loss in a subject suffering from cancer cachexia, the treatable subject is preferably a subject whose weight loss in the past 14 days is 2% or more.

[0056] Early satiety refers to the tendency to experience a feeling of fullness or satiety early after ingesting food.

[0057] Fatigue is often defined as feeling of weariness, tiredness, or lack of energy. Fatigue can also be defined from a patient-rated perspective involving various assessments or self-assessments, including questions designed to rank feelings of weariness, tiredness, or lack of energy. Specific assessments include the 27-item Functional Assessment of Cancer Therapy-General (FACT-G) and the FACIT-F, which contains a Fatigue Subscale consisting of 13 questions, scored 0-4, and measures patient perceptions of concerns related to fatigue and anemia.

[0058] <Improvement of symptoms of cancer cachexia>

[0059] In the present invention, even among the symptoms of cancer cachexia, it is particularly preferred to improve weight loss, low nutritional status and anemia. It is required to improve the QOL and ADL during survival by improving these symptoms. In particular, when the subject with cancer cachexia is in the late stage of cancer, it is preferred to suppress, alleviate or control weight loss, low nutritional status and anemia before death, and to face death peacefully.

[0060] The following is an explanation of the mechanism of action of the cancer cachexia improving agent.

[0061] <Mechanism of action of agents that improve cancer cachexia>

[0062] (Suppression of cytokines in blood)

[0063] By inhibiting cytokines in the blood, weight loss, anemia and low nutritional status can be improved even in the symptoms of cachexia.

[0064] Cytokines refer to the general term for proteins involved in inflammatory responses and immune responses, and more than hundreds of them are known. Each cytokine has a variety of activities, and they interact in a balanced and coordinated manner to maintain or regulate biological functions. Usually, cytokines are produced / released by immune responses to infections, but in cancer cachexia, the production / release of cytokines by tumor cells is disordered, and the concentrations of multiple cytokines in the blood will rise abnormally. In particular, inflammatory cytokines are involved in the pathological state of cancer cachexia, especially IL-1, IL-6, TNFα, and IFN-γ. In addition, as examples of cytokines that should be inhibited, IL-2, IL-4, IL-10, IL-17, IL-18, MIG, MIP-1α, etc. can be listed. Cytokines released by tumor cells activate the host's immune / inflammatory cells by promoting local inflammatory responses in the tumor, causing the so-called cytokine storm. In cancer cachexia, the pathological state of weight loss caused by skeletal muscle loss is characteristic.

[0065] IL-1 is an inflammatory cytokine involved in defense against inflammation or infection.

[0066] IL-2 is a cytokine involved in cellular immunity. Overexpression of IL-2 is related to the selective increase of regulatory T cells (Treg), which is a subpopulation of T cells. Treg plays a role in maintaining peripheral tolerance by suppressing the immune response of other cells. It is believed that the flaws in peripheral tolerance will cause autoimmune diseases in humans. Therefore, it is believed that the immunosuppressive ability of Treg can prevent the onset of autoimmune diseases. In addition, Treg is also related to cancer, and solid tumors and hematological malignancies are accompanied by an increase in the number of Tregs (see

[0007] of Japanese Patent Gazette No. 2020-002154).

[0067] IL-4 is a non-repetitive cytokine involved in the differentiation of helper T cells into Th2 (helper T2 type) subsets, and Th2 promotes the differentiation of immature B cells into plasma cells that produce IgE. IgE levels rise in allergic asthma. Therefore, IL-4 is related to the onset of allergic asthma (see

[0008] of Japanese Patent Publication No. 2020-002154).

[0068] IL-6 is a B cell differentiation factor, B cell stimulating factor-2, hepatocyte stimulating factor, hybridoma growth factor, and plasmacytoma growth factor. IL-6 is a representative inflammatory cytokine. In addition, IL-6 is also a multifunctional cytokine involved in the control of acute inflammatory response, regulation of specific immune responses including B and T cell differentiation, bone metabolism, platelet production, epithelial proliferation, menstruation, neural cell differentiation, neuroprotection, age increase, cancer, inflammatory response occurring in Alzheimer's disease, etc. IL-6 is believed to play a role in the development of various diseases and disorders including fatigue, cachexia, autoimmune diseases, skeletal system diseases, cancer, heart disease, obesity, diabetes, asthma, Alzheimer's disease and multiple sclerosis (refer to

[0002] to

[0005] of Japanese Patent Publication No. 2019-047787).

[0069] IL-10 is a homodimeric protein of 160 amino acid residues produced in T cells, macrophages, and dendritic cells. IL-10 is known to be involved in macrophage function inhibition, B cell activation, etc. (see

[0052] of Japanese Patent Application No. 2018 / 212237). In addition, IL-10 is an anti-inflammatory cytokine.

[0070] IL-17 is a protein of 132 amino acid residues produced in CD4 memory T cells and Th17 cells. IL-17 is known to be involved in the production of inflammatory cytokines by macrophages, epithelial cells, endothelial cells, fibroblasts, etc. (see

[0053] of Japanese Patent Application No. 2018 / 212237).

[0071] When cells are subjected to strong stress due to viral infection or cancer reactive oxygen species, IL-18 is released.

[0072] IFN (interferon) has the function of inhibiting viral replication and activating immune cells (such as natural killer cells and macrophages). IFN is divided into type I IFN, type II IFN and type III IFN. Among them, IFNγ, which is a type II IFN, is a multifaceted cytokine produced by activated immune cells, which can induce cellular responses such as increased macrophage activity, increased MHC molecule expression and increased NK cell activity (see

[0209] of Japanese Patent Application No. 2020-522254).

[0073] TNF (Tumor Necrosis Factor) is a representative inflammatory cytokine. As TNF, TNFα, TNFβ and LTβ are known. Among them, TNFα mediates several important physiological functions including the organization of the structure and function of secondary lymphoid organs, apoptosis and anti-tumor activity, inhibition of viral replication, immunoregulation and inflammation. In addition, TNF also plays an important role in the pathogenesis of autoimmune diseases, acute phase reactions, septic shock, fever and cachexia (see

[0004] of Japanese Patent Gazette No. 2020-079306).

[0074] MIG is a cytokine that controls type 1 immune response. It is produced by macrophages, epithelial cells, endothelial cells, etc., and causes Th1 cells, CD8T cells, and some macrophages to migrate to the site of inflammation.

[0075] MIP-1α is a cytokine produced by macrophages, fibroblasts, epithelial cells, vascular smooth muscle cells, and the like in response to inflammation.

[0076] Among these, the agent for improving cancer cachexia of the present invention is preferably used for suppressing an increase in the IL-6 concentration and TFN-α concentration in the blood of a subject suffering from cancer cachexia.

[0077] (Improvement in the amount of blood cells, etc.)

[0078] The cancer cachexia improver of the present invention can preferably improve the amount of blood cells in the blood, and more preferably improve the number of red blood cells, hemoglobin value (hemoglobin number), platelet number, and hematocrit value. The cancer cachexia improver of the present invention is particularly preferably used to inhibit the decrease in the number of red blood cells, hemoglobin value, and hematocrit value of a subject with cancer cachexia. By improving these, low nutritional status and anemia are improved.

[0079] (Type of cancer)

[0080] The cancer cachexia improving agent of the present invention can be implemented in any type of cancer, and these methods are preferably implemented in the type of cancer that is usually accompanied by cancer cachexia. Non-limiting examples of related cancers include, for example, breast cancer, prostate cancer, multiple myeloma, transitional cell carcinoma, lung cancer (e.g., non-small cell lung cancer (NSCLC)), kidney cancer, thyroid cancer and other cancers that cause hyperparathyroidism, adenocarcinoma, leukemia (e.g., chronic myeloid leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, acute lymphocytic leukemia), lymphoma (e.g., B cell lymphoma, T cell lymphoma, non-Hodgkin's lymphoma, Hodgkin lymphoma), head and neck cancer, esophageal cancer, stomach cancer, large intestine cancer, intestinal cancer, colorectal cancer, rectal cancer, pancreatic cancer, liver cancer, bile duct cancer, gallbladder cancer, ovarian cancer, endometrial cancer, vaginal cancer, cervical cancer, bladder cancer, neuroblastoma, sarcoma, osteosarcoma, malignant melanoma, squamous cell carcinoma, including primary bone cancer (e.g., osteosarcoma, chondrosarcoma, Ewing's sarcoma, fibrosarcoma, malignant fibrous histiocytoma, ameloblastoma (Adamanti noma, adamantoma), giant cell tumor and chordoma) and secondary (metastatic) bone cancer, soft tissue sarcoma, basal cell carcinoma, angiosarcoma (Angiosarcoma), hemangiosarcoma (Hemangiosarcoma), myxosarcoma, liposarcoma, osteosarcoma (bone sarcoma), angiosarcoma, endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, testicular cancer, uterine cancer, digestive tract cancer, mesothelioma, leiomyosarcoma , rhabdomyosarcoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, Waldenstroma macroglobulinemia, papillary adenocarcinoma, cystadenocarcinoma, bronchogenic carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, epithelial carcinoma, glioma, glioblastoma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, retinoblastoma, medullary carcinoma, thymoma, etc. In a preferred embodiment, the cancer is non-small cell lung cancer (NSCLC), more preferably unresectable stage III or IV NSCLC.

[0081] <Ingredients of the agent for improving cancer cachexia>

[0082] The active ingredient of the cancer cachexia improving agent of the present invention is the culture supernatant of dental pulp-derived stem cells or microparticles derived from the culture supernatant of dental pulp-derived stem cells.

[0083] Compared with the compositions that can be used as cancer cachexia improving agents in the past, the cancer cachexia improving agent of the present invention has the following advantages: it is easy to mass produce, and the culture medium of stem cells that was previously discarded as industrial waste can be effectively utilized, and the waste cost of the stem cell culture medium can be reduced. In particular, when the culture supernatant of stem cells from dental pulp is the culture supernatant of stem cells from human dental pulp, it also has the following advantages: when the cancer cachexia improving agent of the present invention is applied to humans, it is highly safe from the perspective of immunology and the like, and there are fewer ethical issues. When the culture supernatant of stem cells from dental pulp is the culture supernatant of stem cells from the dental pulp of a patient suffering from cancer cachexia, when the cancer cachexia improving agent of the present invention is applied to the patient, the safety is further improved and the ethical issues are reduced.

[0084] Since the cancer cachexia improver of the present invention is derived from the culture supernatant of stem cells from dental pulp, it is also used for restorative medical purposes. In particular, a composition containing microparticles derived from the culture supernatant of stem cells from dental pulp is preferably used for restorative medical purposes. Here, it is known that in regenerative medicine based on stem cell transplantation, stem cells are not the protagonist of regeneration, but the liquid components produced by stem cells repair organs together with their own stem cells. Difficult problems such as cancerization, standardization, administration method, preservation, and culture method associated with previous stem cell transplantation have been solved, and restorative medical treatment can be performed using a composition using the culture supernatant of stem cells from dental pulp or microparticles thereof. Compared with stem cell transplantation, since there are no transplanted cells when using the cancer cachexia improver of the present invention, it is not easy to cause tumorization, etc., which can be said to be safer. In addition, the cancer cachexia improver of the present invention has the advantage that a certain standardized quality improver can be used. Since mass production or effective administration methods can be selected, it can be used at low cost.

[0085] (Culture supernatant of dental pulp-derived stem cells)

[0086] - Stem cells from dental pulp -

[0087] The culture supernatant of dental pulp-derived stem cells and the like is not particularly limited.

[0088] The culture supernatant of dental pulp-derived stem cells or the like preferably contains substantially no serum. For example, the culture supernatant of dental pulp-derived stem cells or the like preferably has a serum content of 1% by mass or less, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

[0089] Dental pulp-derived stem cells may be derived from humans or animals other than humans. Examples of animals other than humans include the same animals (organism species) as those to which the cancer cachexia ameliorating agent of the present invention is administered, which will be described later, and preferably mammals.

[0090] There is no particular limitation on the dental pulp stem cells used for the culture supernatant. Exfoliated deciduous teeth dental pulp stem cells (Stem Cells From Exfoliated Deciduous Teeth), deciduous teeth dental pulp stem cells obtained by other methods, or permanent teeth dental pulp stem cells (Dental Pulp Stem Cells; DPSC) can be used. In addition to human deciduous teeth dental pulp stem cells or human permanent teeth dental pulp stem cells, dental pulp stem cells derived from animals other than humans, such as pig deciduous teeth dental pulp stem cells, can also be used.

[0091] In addition to exosomes, dental pulp-derived stem cells can produce various cytokines such as vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), insulin-like growth factor (IGF), platelet-derived growth factor (PDGF), transforming growth factor-β (TGF-β)-1 and -3, TGF-α, KGF, HBEGF, SPARC, other growth factors, chemokines, etc. In addition, they can also produce a variety of other physiologically active substances.

[0092] In the present invention, the dental pulp-derived stem cells used in the culture supernatant of dental pulp-derived stem cells are preferably dental pulp-derived stem cells containing a large amount of protein, and deciduous tooth dental pulp stem cells are preferably used. That is, in the present invention, the culture supernatant of deciduous tooth dental pulp stem cells is preferably used.

[0093] The dental pulp-derived stem cells used in the present invention may be natural stem cells or genetically modified stem cells as long as they can achieve the target treatment.

[0094] In particular, immortalized stem cells derived from dental pulp stem cells can be used in the present invention. By using immortalized stem cells that can proliferate indefinitely in essence, the amount and composition of biological factors contained in the culture supernatant of stem cells can be stabilized for a long time. There are no particular restrictions on the immortalized stem cells derived from dental pulp stem cells. The immortalized stem cells are preferably immortalized stem cells that are not cancerous. The immortalized stem cells derived from dental pulp stem cells can be modulated by adding the following small molecule compounds (inhibitors) to the dental pulp stem cells alone or in combination for culturing.

[0095] The TGFβ receptor inhibitor is not particularly limited as long as it is a substance that has the effect of inhibiting the function of transforming growth factor (TGF) β receptor, for example, 2-(5-benzo[1,3]dioxol-4-yl-2-tert-butyl-1H-imidazol-4-yl)-6-methylpyridine, 3-(6-methylpyridin-2-yl)-4-(4-quinolyl)-1-phenylthiocarbamoyl-1H-pyrazole (A- A-83-01), 2-[(5-chloro-2-fluorophenyl)pteridin-4-yl]pyridin-4-ylamine (SD-208), 3-[(pyridin-2-yl)-4-(4-quinone)]-1H-pyrazole, 2-(3-(6-methylpyridin-2-yl)-1H-pyrazol-4-yl)-1,5-naphthyridine (all from Merck), SB431542 (Sigma-Aldrich), etc. Preferably, A-83-01 is mentioned.

[0096] There is no particular limitation on ROCK inhibitors, as long as they are substances that have the effect of inhibiting the function of Rho binding kinase. Examples of ROCK inhibitors include GSK269962A (Axonmedchem), Fasudil Hydrochloride (Tocris Bioscience), Y-27632, H-1152 (all from Fujifilm Wako Pure Chemical Industries, Ltd.), etc. Preferably, Y-27632 is used.

[0097] The GSK3 inhibitor is not particularly limited as long as it is a substance that inhibits GSK-3 (Glycogen Synthase Kinase 3), and examples thereof include A1070722, BIO, and BIO-acetone oxime (all from TOCRIS).

[0098] There is no particular limitation on MEK inhibitors, as long as they are substances that have the effect of inhibiting the function of MEK (MAP kinase-ERK kinase), for example, AZD6244, CI-1040 (PD184352), PD0325901, RDEA119 (BAY86-9766), SL327, U0126-EtOH (all from Selleck), PD98059, U0124, U0125 (all from Cosmo Bio Co., Ltd.), etc.

[0099] When the cancer cachexia improving agent of the present invention is used in regenerative medicine, based on the requirements of the law on safety assurance of regenerative medicine, etc., the composition containing the culture supernatant of dental pulp-derived stem cells or immortalized stem cells thereof or microparticles derived therefrom is a configuration that does not contain other somatic stem cells other than dental pulp-derived stem cells, etc. The cancer cachexia improving agent of the present invention may contain mesenchymal stem cells or other somatic stem cells other than dental pulp-derived stem cells, etc., but preferably does not contain them.

[0100] Examples of somatic stem cells other than mesenchymal stem cells include stem cells from the dermis, digestive system, bone marrow, nervous system, etc., but are not limited to these. Examples of somatic stem cells of the dermis include epithelial stem cells, hair follicle stem cells, etc. Examples of somatic stem cells of the digestive system include pancreatic (whole) stem cells, liver stem cells, etc. Examples of somatic stem cells of the bone marrow (other than mesenchymal stem cells) include hematopoietic stem cells, etc. Examples of somatic stem cells of the nervous system include neural stem cells, retinal stem cells, etc.

[0101] The cancer cachexia improving agent of the present invention may contain stem cells other than somatic stem cells, but preferably does not contain any. Stem cells other than somatic stem cells include embryonic stem cells (ES cells), induced pluripotent stem cells (iPS cells), and embryonic carcinoma cells (EC cells).

[0102] -Method for preparing culture supernatant of dental pulp-derived stem cells-

[0103] The method for preparing the culture supernatant of dental pulp-derived stem cells or immortalized stem cells thereof is not particularly limited, and conventional methods can be employed.

[0104] The culture supernatant of stem cells from dental pulp, etc. is a culture solution obtained by culturing stem cells from dental pulp. For example, by separating and removing cell components after culturing stem cells from dental pulp, a culture supernatant that can be used in the present invention can be obtained. A culture supernatant that has been appropriately subjected to various treatments (e.g., centrifugal treatment, concentration, solvent replacement, dialysis, freezing, drying, freeze drying, dilution, desalting, preservation, etc.) can be used. In particular, the culture supernatant is preferably a culture supernatant containing microparticles obtained by subjecting the culture solution obtained by culturing stem cells from dental pulp to a centrifugal treatment to remove stem cells from dental pulp or their dead cells. After the centrifugal treatment, a specific component containing a large number of microparticles can be separated to modulate the culture supernatant.

[0105] The dental pulp stem cells used to obtain the culture supernatant of dental pulp stem cells can be screened by conventional methods, and can be screened based on the size or morphology of the cells, or as adhesive cells. The dental pulp cells collected from the deciduous teeth or permanent teeth can be screened as adhesive cells or their passage cells. In the culture supernatant of dental pulp stem cells, the culture supernatant obtained by culturing the selected stem cells can be used.

[0106] It should be noted that the "culture supernatant of dental pulp-derived stem cells, etc." is preferably a culture solution obtained by culturing dental pulp-derived stem cells, etc., and does not contain the cells themselves. The culture supernatant of dental pulp-derived stem cells used in the present invention preferably does not contain cells as a whole (regardless of the type of cells) in one embodiment thereof. The composition of this embodiment naturally distinguishes the dental pulp-derived stem cells themselves from various compositions containing dental pulp-derived stem cells based on this feature. A typical example of this embodiment is a composition that does not contain dental pulp-derived stem cells and consists only of the culture supernatant of dental pulp-derived stem cells.

[0107] The culture supernatant of dental pulp-derived stem cells used in the present invention may contain the culture supernatants of both stem cells from deciduous tooth pulp and stem cells from adult dental pulp. The culture supernatant of dental pulp-derived stem cells used in the present invention preferably contains the culture supernatant of stem cells from deciduous tooth pulp as an effective ingredient, more preferably contains 50% by mass or more, and preferably contains 90% by mass or more. The culture supernatant of dental pulp-derived stem cells used in the present invention is more preferably a composition consisting only of the culture supernatant of stem cells from deciduous tooth pulp.

[0108] In the culture medium for obtaining the culture supernatant of dental pulp stem cells, a basal medium or a culture medium in which serum or the like is added to the basal medium can be used. It should be noted that, as the basal medium, in addition to Dulbecco's modified Eagle's medium (DMEM), Iscove's modified Dulbecco's medium (IMDM) (GIBCO, etc.), HamF-12 medium (HamF12) (SIGMA, GIBCO, etc.), RPMI1640 medium, etc. can also be used. In addition, as examples of components that can be added to the culture medium, serum (fetal bovine serum, human serum, sheep serum, etc.), serum substitutes (Knockout Serum Replacement (KSR: knockout serum substitute)), bovine serum albumin (BSA), antibiotics, various vitamins, and various minerals can be listed.

[0109] However, in order to prepare a "culture supernatant of dental pulp-derived stem cells" that does not contain serum, a serum-free medium can be used for the subculture throughout the entire process or at the end or for several times since the end. For example, by culturing dental pulp-derived stem cells with a serum-free medium (serum-free medium), a culture supernatant of dental pulp-derived stem cells that does not contain serum can be prepared. By performing one or more subcultures, and culturing the last or several times since the end with a serum-free medium, a culture supernatant of dental pulp-derived stem cells that does not contain serum can also be obtained. On the other hand, by removing serum from the recovered culture supernatant using dialysis or column-based solvent replacement, a culture supernatant of dental pulp-derived stem cells that does not contain serum can also be obtained.

[0110] In the culture of dental pulp-derived stem cells for obtaining the culture supernatant, the conditions commonly used can be directly applied. The method for preparing the culture supernatant of dental pulp-derived stem cells can be carried out in the same manner as the cell culture method described below, except that the steps of separation and selection of stem cells are appropriately adjusted according to the type of stem cells. A person skilled in the art can appropriately separate and select dental pulp-derived stem cells according to the type of dental pulp-derived stem cells.

[0111] In addition, in order to mass-produce exosomes in the culture of dental pulp-derived stem cells, special conditions may be applied, such as co-culture conditions with any stimulant, such as low temperature conditions, low oxygen conditions, and microgravity conditions.

[0112] In the present invention, the culture of dental pulp-derived stem cells used for preparing exosomes may contain other components in addition to the culture supernatant containing dental pulp-derived stem cells, but preferably does not substantially contain other components.

[0113] However, various additives for preparing exosomes may be added to the culture supernatant of dental pulp-derived stem cells before storage.

[0114] (Micro particles)

[0115] The microparticles are derived from the dental pulp-derived stem cells, for example, by secretion, budding, or dispersion from the dental pulp-derived stem cells, and are leached, released, or shed into the cell culture medium. Therefore, the microparticles are contained in the culture supernatant of the dental pulp-derived stem cells.

[0116] The microparticles derived from the culture supernatant of dental pulp-derived stem cells may be used in a state contained in the culture supernatant or in a state purified from the culture supernatant. The microparticles are preferably microparticles purified from the culture supernatant.

[0117] The source of microparticles can be determined by known methods. For example, the method described in J Stem Cell Res Ther (2018) 8:2 can be used to determine whether the microparticles are derived from dental pulp stem cells, adipose stem cells, bone marrow stem cells, umbilical cord stem cells, etc. Specifically, the source of each microparticle can be determined based on the miRNA pattern of the microparticles.

[0118] The microparticles are preferably at least one selected from exosomes, microvesicles, membrane particles, membrane vesicles, ectosomes, exovesicles, or microvesicles, and are more preferably exosomes.

[0119] The diameter of the fine particles is preferably 10 to 1000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm.

[0120] In addition, it is desirable that molecules called tetraspanins such as CD9, CD63, and CD81 exist on the surface of the microparticles. These molecules may be CD9 alone, CD63 alone, CD81 alone, or any combination of two or three of them.

[0121] Hereinafter, a preferred embodiment in the case of using exosomes as microparticles will be described in some cases, but the microparticles used in the present invention are not limited to exosomes.

[0122] Exosomes are preferably extracellular vesicles released from cells upon fusion of multicellular bodies with the plasma membrane.

[0123] The surface of the exosome preferably contains lipids and proteins derived from the cell membrane of dental pulp-derived stem cells.

[0124] The interior of the exosome preferably contains intracellular substances derived from dental pulp stem cells, such as nucleic acids (microRNA, messenger RNA, DNA, etc.) and proteins.

[0125] Exosomes are known to be used for cell-to-cell communication by transporting genetic information from one cell to other cells. Exosomes can be easily tracked and can be targeted to specific areas.

[0126] - Content of microparticles -

[0127] The cancer cachexia improving agent of the present invention has no particular limitation on the content of the microparticles. The cancer cachexia improving agent of the present invention preferably contains 0.5×10 8 More than 1.0×10 8More than 2.0×10 8 More preferably, it contains 2.5×10 8 More preferably, it contains 1.0×10 9 More than one.

[0128] In addition, the cancer cachexia improving agent of the present invention has no particular limitation on the concentration of the microparticles contained. The cancer cachexia improving agent of the present invention preferably contains 1.0×10 8 More than 2.0×10 8 pcs / mL or more, particularly preferably 4.0×10 8 More preferably, it contains 5.0×10 8 More preferably, it contains 2.0×10 9 More than pcs / mL.

[0129] The preferred embodiment of the cancer cachexia ameliorating agent of the present invention contains microparticles in such a large amount or at a high concentration, so that the amount of blood cells in the blood can be maintained at a high level, the amount of (especially inflammatory) cytokines can be suppressed, and there is a tendency to extend the survival period to a certain extent.

[0130] - Method for preparing micro particles -

[0131] Microparticles can be prepared by purifying microparticles from the culture supernatant of dental pulp-derived stem cells or the like.

[0132] The purification of the microparticles is preferably performed by separating a fraction containing the microparticles from the culture supernatant of the dental pulp-derived stem cells, and more preferably by isolating the microparticles.

[0133] Microparticles can be separated from non-associated components by separation based on the properties of the microparticles. For example, microparticles can be separated based on molecular weight, size, morphology, composition or biological activity.

[0134] In the present invention, the culture supernatant of stem cells from dental pulp can be centrifuged and the obtained specific component (e.g., precipitate) containing a large number of microparticles can be separated to purify the microparticles. Unnecessary components (insoluble components) of components other than the specified components can be removed. The removal of the solvent and dispersion medium, as well as unnecessary components from the cancer cachexia improving agent of the present invention can be incomplete removal. The conditions of the centrifugation treatment can be exemplified as follows: 1 to 30 minutes at 100 to 20,000 g.

[0135] In the present invention, microparticles can be purified by filtering the culture supernatant of stem cells from dental pulp or its centrifugal product. Unnecessary components can be removed by filtering. In addition, if a filter membrane with an appropriate pore size is used, the removal of unnecessary components and sterilization can be performed at the same time. There is no particular limitation on the material, pore size, etc. of the filter membrane used for filtering. Filtration can be performed using a filter membrane with an appropriate molecular weight or size cutoff according to a known method. From the viewpoint of easy separation of exosomes, the pore size of the filter membrane is preferably 10 to 1000 nm, more preferably 30 to 500 nm, and particularly preferably 50 to 150 nm.

[0136] In the present invention, a further separation method such as column chromatography can be used to separate the culture supernatant of dental pulp stem cells or its centrifuged product or its filtered product. For example, high performance liquid chromatography (HPLC) using various columns can be used. The column can use a size exclusion column or a binding column.

[0137] In order to track the microparticles (or their activity) in each component at each processing stage, one or more properties or biological activities of the microparticles can be used. For example, light scattering, refractive index, dynamic light scattering or UV-visible light detectors can be used to track the microparticles. Alternatively, specific enzyme activities can be used to track the activities in each component.

[0138] As a method for purifying fine particles, the method described in

[0034] to

[0064] of JP-A-2019-524824 can be used, and the contents of this publication are incorporated into this specification as a reference.

[0139] <Other ingredients>

[0140] The cancer cachexia improving agent of the present invention may contain other ingredients in addition to the microparticles, depending on the type of animal to be administered or the purpose, within the range that does not impair the effect of the present invention. Other ingredients include: nutrients, antibiotics, cytokines, protective agents, carriers, excipients, disintegrants, buffers, emulsifiers, suspending agents, analgesics, stabilizers, preservatives, preservatives, etc.

[0141] Examples of the nutritional components include fatty acids, vitamins, and the like.

[0142] Examples of the antibiotic include penicillin, streptomycin, and gentamicin.

[0143] Examples of the carrier include materials known as pharmaceutically acceptable carriers.

[0144] The cancer cachexia improving agent of the present invention may be the culture supernatant or microparticles of dental pulp-derived stem cells themselves, or may be a pharmaceutical composition further comprising a pharmaceutically acceptable carrier or excipient, etc. The purpose of the pharmaceutical composition is to facilitate administration of the culture supernatant or microparticles of dental pulp-derived stem cells to the subject.

[0145] The pharmaceutically acceptable carrier is preferably a carrier (including a diluent) that does not cause significant irritation to the subject and does not inhibit the biological activity and properties of the administered compound. Examples of carriers are: propylene glycol; (physiological) saline; emulsion; buffer; culture medium, such as DMEM or RPMI; and cryopreservation medium containing a component that removes free radicals.

[0146] The cancer cachexia improving agent of the present invention may contain the active ingredients of the cancer cachexia improving agent known in the past, and those skilled in the art may make appropriate changes according to the application or the subject of administration.

[0147] On the other hand, the agent for improving cancer cachexia of the present invention preferably does not contain a predetermined substance.

[0148] For example, the agent for improving cancer cachexia of the present invention preferably does not contain stem cells derived from dental pulp.

[0149] In addition, the cancer cachexia improver of the present invention preferably does not contain MCP-1. However, it may contain cytokines other than MCP-1. As other cytokines, cytokines described in

[0014] to

[0020] of Japanese Patent Publication No. 2018-023343 can be listed.

[0150] In addition, the cancer cachexia improving agent of the present invention preferably does not contain Siglec-9. However, it may contain other sialic acid-binding immunoglobulin-like lectins other than Siglec-9.

[0151] It should be noted that the cancer cachexia improving agent of the present invention preferably does not substantially contain serum (fetal bovine serum, human serum, sheep serum, etc.). In addition, the cancer cachexia improving agent of the present invention preferably does not substantially contain conventional serum substitutes such as knockout serum replacement (KSR).

[0152] The cancer cachexia improving agent of the present invention preferably contains the above-mentioned other components in an amount (solid content) of 1% by mass or less, more preferably 0.1% by mass or less, and particularly preferably 0.01% by mass or less.

[0153] <Method for producing an agent for improving cancer cachexia>

[0154] The method for producing the cancer cachexia-improving agent of the present invention is not particularly limited.

[0155] The culture supernatant of stem cells derived from dental pulp can be prepared according to the above method to prepare an agent for improving cancer cachexia. Next, the microparticles can be purified from the culture supernatant of stem cells derived from dental pulp to prepare the agent for improving cancer cachexia of the present invention. Alternatively, the microparticles can be purified from the commercially available culture supernatant of stem cells derived from dental pulp to prepare the agent for improving cancer cachexia of the present invention. Moreover, a composition containing the culture supernatant of stem cells derived from dental pulp (or appropriately purifying the composition) that is to be discarded can also be received, and the microparticles can be purified therefrom to prepare the agent for improving cancer cachexia of the present invention.

[0156] There is no particular limitation on the final form of the agent for improving cancer cachexia of the present invention. For example, the agent for improving cancer cachexia of the present invention may include: a form in which the culture supernatant of stem cells derived from dental pulp is filled into a container; a form in which the microparticles are filled into a container together with a solvent or a dispersion medium; a form in which the microparticles are gelled together with a gel and filled into a container; a form in which the culture supernatant of stem cells derived from dental pulp or the microparticles are frozen and / or dried and solidified to prepare a preparation or filled into a container, etc. As the container, for example, tubes, centrifuge tubes, bags, etc. suitable for cryopreservation can be mentioned. The freezing temperature can be set to, for example, -20°C to -196°C.

[0157] [Method for improving cancer cachexia]

[0158] The method for improving cancer cachexia of the present invention includes: administering an effective amount of the agent for improving cancer cachexia of the present invention to a subject suffering from cancer cachexia.

[0159] There is no particular limitation on the step of administering the agent for improving cancer cachexia of the present invention to a subject suffering from cancer cachexia.

[0160] Examples of the administration method include: spraying or suction into the oral cavity, nasal cavity or respiratory tract, drip infusion, topical administration, nasal drops, etc., and less invasiveness is preferred. As the method of topical administration, injection is preferred. In addition, electroporation is also preferred, which is to apply a voltage (electrical pulse) to the skin surface to temporarily create fine pores on the cell membrane, so that the active ingredient penetrates into the dermis layer that cannot be reached by conventional care. In the case of topical administration, intravenous administration, intraarterial administration, intraportal vein administration, intradermal administration, subcutaneous administration, intramuscular administration, intraperitoneal administration, etc. can be mentioned, and intraarterial administration, intravenous administration, subcutaneous administration or intraperitoneal administration are more preferred.

[0161] The agent for improving cancer cachexia of the present invention administered to a subject suffering from cancer cachexia can circulate in the subject's body and reach the specified tissue.

[0162] There is no particular restriction on the number of administrations and the interval between administrations. The number of administrations can be set to be once or more per week, preferably 5 or more times, more preferably 6 or more times, and particularly preferably 7 or more times. The interval between administrations is preferably 1 hour to 1 week, more preferably half a day to 1 week, and particularly preferably 1 day (once a day). However, it can be appropriately adjusted according to the biological species of the subject or the symptoms of the subject.

[0163] The cancer cachexia improver of the present invention is preferably used to administer the cancer cachexia improver to a subject with cancer cachexia at least once a week during the effective treatment period. When the subject is a human, the administration frequency is preferably high, preferably 5 times a week or more during the effective treatment period, preferably daily.

[0164] The concentration used was 2.0×10 9 In the case of a culture supernatant of dental pulp-derived stem cells at a concentration of cells / ml, in a mouse model, the amount is preferably 0.1 to 5 ml, more preferably 0.3 to 3 ml, and particularly preferably 0.5 to 1 ml per mouse (about 25 g).

[0165] The concentration used was 0.1×10 8 In the case of microparticles of 1 μg / μg, in a mouse model, the dosage is preferably 1 to 50 μg per mouse (about 25 g), more preferably 3 to 30 μg, and particularly preferably 5 to 25 μg.

[0166] The preferred range of the dosage per unit body weight for other animals can be calculated from the dosage per unit body weight (about 25 g) for model mice using a proportional relationship, but can be appropriately adjusted depending on the symptoms of the subject.

[0167] There is no particular limitation on the animal (biological species) to which the cancer cachexia improving agent of the present invention is administered. The animal to which the cancer cachexia improving agent of the present invention is administered is preferably a mammal, a bird (chicken, quail, duck, etc.), or a fish (salmon, trout, tuna, bonito, etc.). As a mammal, it can be a human or a non-human mammal, but preferably a human. As a non-human mammal, it is more preferably a cow, a pig, a horse, a goat, a sheep, a monkey, a dog, a cat, a mouse, a rat, a guinea pig, or a hamster.

[0168] The cancer cachexia improving agent of the present invention can be used in combination with a conventionally known cancer cachexia improving agent.

[0169] Example

[0170] Below, enumerate embodiment and comparative example or reference example, to further specifically illustrate the feature of the present invention.As long as the material, usage amount, ratio, processing content, processing sequence etc. shown in the following embodiment do not depart from the purpose of the present invention, can be appropriately changed.Therefore, the scope of the present invention should not be interpreted restrictively by the specific example shown below.

[0171] [Example 1]

[0172] <Experimental plan>

[0173] according to Figure 1 In the experimental scheme shown, samples were administered to cancer cachexia model mice, followed by whole blood collection to evaluate the improvement of cachexia.

[0174] <Preparation of lethal cancer cachexia model mice>

[0175] For 7-week-old nude mice (Balb / c-nu / nu) used as normal mice, 1×10 6 The lethal cancer cachexia model mice were conditioned with B16-F10-luc-G5 cells (B16-luc cells), which were harsh conditions that caused all mice to die within 18 days.

[0176] <Preparation of culture supernatant of dental pulp-derived stem cells>

[0177] DMEM medium was used instead of DMEM / HamF12 mixed medium, and the culture supernatant of human deciduous dental pulp stem cells was prepared according to the method described in Example 6 of Japanese Patent No. 6296622, and the culture supernatant was separated. In the primary culture, fetal bovine serum (FBS) was added for culture, and in the subculture, the supernatant of the subculture medium cultured with the primary culture medium was separated to make it free of FBS, and the culture supernatant of deciduous dental pulp stem cells was prepared. It should be noted that DMEM is Dulbecco's modified Eagle medium, and F12 is HamF-12 medium.

[0178] The culture supernatant of the prepared dental pulp-derived stem cells was used as a sample of the agent for improving cancer cachexia in Example 1.

[0179] <Provision of samples>

[0180] On the day of treatment (day 0) of preparing cancer cachexia model mice and 7 and 14 days after the treatment, 0.5 ml (1.0×10 9 ) were given the sample of Example 1.

[0181] [Example 2]

[0182] <Exosome Modulation>

[0183] Exosomes derived from dental pulp stem cells were purified from the culture supernatant of the dental pulp stem cells obtained in Example 1 according to the following method.

[0184] After filtering the culture supernatant (100 mL) of deciduous dental pulp stem cells with a filter having a pore size of 0.22 μm, the solution was centrifuged at 100,000 × g for 60 minutes at 4°C. The supernatant was decanted and the exosome concentrated particles were resuspended in phosphate buffered saline (PBS). The resuspended sample was centrifuged at 100,000 × g for 60 minutes. The particles were recovered from the bottom of the centrifuge tube as a concentrated sample again (about 100 μl). The protein concentration was determined by a micro BSA protein assay kit (Pierce, Rockford, IL). The composition containing exosomes (concentrated solution) was stored at -80°C.

[0185] As the sample of the agent for improving cancer cachexia in Example 2, a composition containing exosomes purified from the culture supernatant of dental pulp-derived stem cells was used.

[0186] The average particle size and concentration of the fine particles contained in the cancer cachexia ameliorating agent of each example were evaluated.

[0187] The average particle size of the fine particles contained in the cancer cachexia improving agent of each example is 50 to 150 nm.

[0188] The improving agent for cancer cachexia in each example is 1.0×10 9 The high concentration of exosome solution above 10 / ml, especially the improving agent for cancer cachexia of Example 1, is 2.0×10 9 / ml high concentration exosome solution.

[0189] In addition, the components of the cancer cachexia improving agent obtained in each embodiment were analyzed according to a known method. As a result, it can be seen that the cancer cachexia improving agent in each embodiment does not contain stem cells from dental pulp stem cells, MCP-1, or Siglec-9. Therefore, it can be seen that the active ingredients of MCP-1 and Siglec-9 and their analogs, which are different from the active ingredients of the culture supernatant of mesenchymal stem cells, are the active ingredients of the cancer cachexia improving agent in each embodiment.

[0190] In addition to administering 20 μg (2.0×10 8 The sample was administered in the same manner as in Example 1 except that the sample of Example 2 was used instead of the sample of Example 1.

[0191] [Comparative Example 1]

[0192] The sample was administered in the same manner as in Example 1, except that an equal amount of 0.5 ml of physiological saline as the sample of Comparative Example 1 was administered to the cancer cachexia model mice prepared in Example 1 instead of the sample of Example 1.

[0193] [evaluate]

[0194] (Evaluation of weight loss)

[0195] The body weight of the cancer cachexia model mice was measured on the treatment day (day 0) and 7, 14, and 21 days after the treatment day. Each group had n=10, and for example, in Example 1, the average body weight of 10 mice administered with the sample of Example 1 was determined.

[0196] 7-week-old nude mice (Balb / c-nu / nu) were used as a reference example of normal mice. The weight of the normal mice in Reference Example 1 was used as the control (100%), and the weights of 10 mice given the samples of each embodiment and comparative example were calculated (relative to the % of the control). The results are shown in Figure 2 .

[0197] Depend on Figure 2 The results showed that administration of the cancer cachexia improving agent of the present invention to cancer cachexia model mice can suppress body weight loss compared to administration of physiological saline.

[0198] From these results, it is predicted that the cancer cachexia improving agent of the present invention can maintain skeletal muscle mass by suppressing weight loss in cachexia patients, improve anemia, and improve QOL and ADL.

[0199] It should be noted that, based on the results of the Examples described below, the mechanism of action for suppressing weight loss is believed to be the suppression of inflammatory cytokines or the suppression of cytokine storms.

[0200] (Evaluation of blood cells, etc.)

[0201] Whole blood was collected 21 days after the treatment day (day 0) of the cancer cachexia model mice. Then, the number of red blood cells (unit: 10,000 / μL), hemoglobin value (hemoglobin. Unit: g / dL), platelet count (unit: 10,000 / μL), and hematocrit value (%) in the blood were quantified. The results are shown in Table 1.

[0202] The results in Table 1 show that by administering the cancer cachexia improving agent of the present invention to cancer cachexia model mice, the red blood cell count, hemoglobin value (hemoglobin), platelet count and hematocrit value can be maintained at high levels compared to the case of administering physiological saline.

[0203] In addition, anemia-like symptoms were observed in the cancer cachexia model mice of Comparative Example 1 to which physiological saline was administered, but anemia-like symptoms were particularly not observed in the cancer cachexia model mice to which the cancer cachexia-improving agent of the present invention was administered.

[0204] From these results, it is predicted that the agent for improving cancer cachexia of the present invention can improve QOL and ADL by improving anemia in cachexia patients.

[0205] [Table 1]

[0206]

[0207] (Evaluation of cytokine levels in blood)

[0208] Whole blood was collected 21 days after the treatment day (day 0) of the cancer cachexia model mice. Then, IL-6 and TNF-α in the blood were quantified using Immune Monitoring 48-Plex Mouse ProcartaPlex (registered trademark) Panel (manufactured by Invitrogen). At the same time, whole blood was collected from normal mice as reference example 1, and the cytokines contained in the blood were quantified in the same manner. The results are shown in Figure 3 and Figure 4 .

[0209] Depend on Figure 3 and Figure 4 The results showed that administration of the cancer cachexia improving agent of the present invention to cancer cachexia model mice could suppress increases in blood IL-6 and TNF-α concentrations compared to administration of physiological saline.

[0210] These results predict that the cancer cachexia improving agent of the present invention can increase appetite by inhibiting the production of inflammatory cytokines in cachexia patients, or improve metabolic abnormalities by inhibiting local inflammatory reactions in tumors or inhibiting cytokine storms, thereby improving QOL and ADL.

[0211] (Evaluation of survival rate in a lethal cancer cachexia mouse model (1))

[0212] From the day of treatment (day 0) of the cancer cachexia model mice, the survival rate of mice in each group (n=10) was confirmed every day. Figure 5 .

[0213] Depend on Figure 5 The results showed that when the cancer cachexia improving agent of the present invention was administered to cancer cachexia model mice at the exosome concentrations of Examples 1 and 2, the average survival days were slightly prolonged and the survival rate was slightly improved compared to the administration of physiological saline.

[0214] [Example 11]

[0215] (Evaluation of survival rate in a lethal cancer cachexia mouse model (2))

[0216] In addition to administering 25 μg (2.5×10 8 The survival rate of Example 11 was evaluated in the same manner as in Example 2 except that the sample of Example 2 (exosomes purified from the culture supernatant of stem cells from dental pulp) was used. It should be noted that the sample administration amount of Example 11 was 1.25 times the sample administration amount of Example 2. Example 11 was performed with n=10. The results are shown in Figure 6 .

[0217] Depend on Figure 6 The results showed that when the cancer cachexia model mice were given the cancer cachexia improving agent of the present invention at an exosome concentration increased to that of Example 11, the average survival days were prolonged and the survival rate was improved compared to the case of administering physiological saline.

[0218] [Example 21]

[0219] <Confirmation of inhibition of cytokine storm in vitro>

[0220] In order to strengthen the evaluation results of the amount of cytokines in the blood in Example 2, a cell model in which cytokine storm is induced was used instead of the cancer cachexia model mouse to confirm the cytokine storm inhibitory effect of the cancer cachexia ameliorating agent of the present invention.

[0221] The cytokine storm was induced using human peripheral blood mononuclear cells (PBMCs), and the cytokine storm suppressive effect of the sample of Example 2 was confirmed in vitro according to the following method.

[0222] Human PBMCs were cultured and stimulated with concanavalin A (Con-A, manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.), a cytokine storm inducer, to induce a cytokine storm.

[0223] The sample of Example 2 and one of the physiological saline solutions as a control were added to the system for treatment, and the cytokines contained in the culture supernatant of human PBMC were quantified using a cytokine assay ELISA kit (manufactured by R&D Systems, trade name Quantikine ELISA Kit). The quantified cytokines were IL-2, IL-4, IL-6, IL-10, IL-17, IFNγ, and TNFα.

[0224] The sample of Example 2 was added so that 100 particles per PBMC cell were treated. After treatment with the sample of Example 2, the amount of cytokines was measured 48 hours later (N=6).

[0225] The results are shown in Table 2 below.

[0226] [Table 2]

[0227]

[0228] (N--6)

[0229] [Example 22]

[0230] <Confirmation of suppression of cytokine storm in vivo>

[0231] The induction of cytokine storm in a mouse model was performed, and the cytokine storm suppressive effect of the sample of Example 2 was confirmed in vivo according to the following method.

[0232] Six 8-week-old C57BL / 6J mice were used in each group.

[0233] Lipopolysaccharide (LPS), a cytokine storm inducer, was administered intraperitoneally to each mouse at 20 mg / kg body weight. It should be noted that cytokine storm can be induced by administering lipopolysaccharide (LPS, endotoxin), which causes symptoms of sepsis such as increased concentrations of inflammatory cytokines (Medical Hypotheses (2020) 144, 109865).

[0234] Immediately after the administration of lipopolysaccharide, an equal amount of 10 μg of the sample of Example 2 (purified by ultracentrifugation) and a control of physiological saline were administered through the tail vein of each mouse. It should be noted that the sample administration amount of Example 21 was 0.5 times the sample administration amount of Example 2.

[0235] Whole blood was collected after 24 hours, and the serum in the cytokine storm of mice was separated. The cytokines contained in the serum of the cytokine storm of mice were quantified using ImmuneMonitoring 48-Plex Mouse ProcartaPlex (registered trademark) Panel (manufactured by Invitrogen). At the same time, whole blood was also collected from normal mice before lipopolysaccharide administration, and the cytokines contained in the serum were quantified in the same way. The quantified cytokines were IL-2, IL-4, IL-6, IL-10, IL-17, IFNγ and TNFα.

[0236] The results are shown in Table 3 below.

[0237] [Table 3]

[0238]

[0239] ND: Not Detected

[0240] (N-6)

[0241] As shown in Tables 2 and 3 above, by administering the sample of Example 2 as the cancer cachexia improving agent of the present invention to animals (mice) in which the cytokine storm is induced and the amount of each cytokine increases, the amount of cytokines in the blood and / or serum, and cytokines other than IL-6 and TNFα are also significantly suppressed compared to the control administered with normal saline, and the cytokine storm can be suppressed. From these results, it is predicted that the cancer cachexia improving agent of the present invention can improve weight loss, anemia, and low nutritional status by inhibiting the inflammatory cytokines or cytokine storm of cachexia patients to increase appetite and improve metabolic abnormalities, thereby improving QOL and ADL.

[0242] [Comparative Examples A to D]

[0243] <Comparison between the culture supernatant and exosomes of dental pulp-derived stem cells in Examples 1 and 2 and the culture supernatant or exosomes of umbilical cord-derived stem cells>

[0244] The researchers investigated whether the production of IL-6 from activated macrophages, which is the main cause of cytokine storm, could be suppressed by adding stem cell culture supernatant or exosomes after activation of mouse macrophages RAW264.7 by stimulation with LPS.

[0245] DMEM and 10% FBS (Catalog No.: DSDSM101) were used as the culture medium. RAW264.7 (1.2×10 6 / mL, TIB-71, purchased from ATCC), cultured in culture medium, added LPS (100ng / mL, ALX-581-007-L002, manufactured by Cosmo Bio Co., Ltd.) and the improving agent samples of cancer cachexia of each example and each comparative example at the time point of reaching 90% subconfluent, and cultured for 18 hours at 37°C and 5% CO2. The amount of IL-6 released into the culture medium after culture was quantified using a mouse IL-6 assay ELISA kit (KE10007, manufactured by Cosmo Bio Co., Ltd.). The obtained measured value (average value of N=2) is shown in Figure 7 .

[0246] In addition, the same test was performed using untreated cells not given LPS (Comparative Example A) and cells given only LPS (Comparative Example B) as controls without adding the cancer cachexia improving agent sample. The results were also as follows: Figure 7 .

[0247] As samples of the cancer cachexia-improving agent, the cancer cachexia-improving agent (stem cell culture supernatant from dental pulp) used in Example 1 of the present specification, the cancer cachexia-improving agent (stem cell exosome from dental pulp) used in Example 2 of the present specification, the stem cell culture supernatant from umbilical cord as Comparative Example C, and the exosome (stem cell exosome from umbilical cord) of Comparative Example D prepared from the stem cell culture supernatant from umbilical cord according to Example 2 were used. Each exosome was separated by ultracentrifugation, and 1,000 exosomes were used per mouse-derived macrophage.

[0248] according to Figure 7 Compared with the case where the culture supernatant of stem cells from the umbilical cord was used (Comparative Example C), the cancer cachexia improving agent of Example 1 using the culture supernatant of stem cells from the dental pulp significantly showed a favorable effect in the cytokine suppression of cells in vitro that induced a cytokine storm. In addition, compared with the case where the microparticles (exosomes) obtained from the culture supernatant of stem cells from the umbilical cord were used (Comparative Example D), the cancer cachexia improving agent of Example 2 using the microparticles (exosomes) obtained from the culture supernatant of stem cells from the dental pulp significantly showed a favorable effect in the cytokine suppression of cells in vitro that induced a cytokine storm.

[0249] Here, in vitro Example 21 is to strengthen the evaluation results of the suppression of the amount of cytokines (IL-6, etc.) in the blood of cancer cachexia model mice in vivo in Example 2. Animal cells that induce cytokine storms are used instead of cancer cachexia model mice to confirm the cytokine storm suppression effect of the cancer cachexia improving agent of the present invention in vitro. That is, by evaluating the suppression of cytokines such as IL-6 in in vitro cells that induce cytokine storms, the improvement effect of cancer cachexia in animals in vivo such as cancer cachexia model mice (suppression of inflammatory cytokines or cytokine storms) can be evaluated, and in addition, appetite can be increased, metabolic abnormalities can be improved, and weight loss, anemia, and low nutritional status can be improved, and QOL and ADL can be improved.

[0250] Therefore, from the results of the comparative test between Example 1 or 2 and Comparative Examples A to D (evaluation results of the inhibition of cytokines such as IL-6 on in vitro cells that induced cytokine storm), it can be seen that the culture supernatant of stem cells from dental pulp or the exosomes obtained therefrom have a significant effect on improving cancer cachexia compared with the culture supernatant of stem cells from umbilical cord or the exosomes obtained therefrom.

Claims

1. Use of a culture supernatant of dental pulp stem cells for producing an agent for improving cancer cachexia, wherein the agent for improving cancer cachexia is an agent for improving cancer cachexia to be administered to a subject suffering from cancer cachexia, and The cancer cachexia improving agent is a composition comprising a culture supernatant of stem cells derived from dental pulp, The culture supernatant contains exosomes derived from the dental pulp-derived stem cells.

2. Use of exosomes derived from the culture supernatant of dental pulp stem cells for the manufacture of an agent for improving cancer cachexia, wherein the agent for improving cancer cachexia is an agent for improving cancer cachexia to be administered to a subject suffering from cancer cachexia, and The agent for improving cancer cachexia is a composition comprising exosomes derived from the culture supernatant of dental pulp-derived stem cells.

3. The use according to claim 1 or 2, which is used for treating weight loss in a subject suffering from cancer cachexia.

4. The use according to claim 3, wherein: The subject has experienced a weight loss of more than 2% in the past 14 days.

5. The use according to claim 1 or 2, which is used for treating anemia in a subject suffering from cancer cachexia.

6. The use according to claim 5, which is used for suppressing the decrease in red blood cell count, hemoglobin value and hematocrit value in a subject suffering from cancer cachexia.

7. The use according to claim 1 or 2, which is used for suppressing the increase of IL-6 concentration and TFN-α concentration in the blood of a subject suffering from cancer cachexia.

8. The use according to claim 1 or 2, which is used for administering the cancer cachexia improving agent to the subject suffering from cancer cachexia at least once a week during the effective treatment period.

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