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Definitions

• the present invention relates to an evaluation system capable of predicting the possibility of developing a drug-induced disorder of the liver and other organs (for example, drug-induced liver disorder).
• Drug-induced liver injury is a side effect that can occur in the process of continuing drug therapy. Drug-induced liver injury is classified into an addictive type, an allergic idiosyncratic type, and a metabolic idiosyncratic type according to the pathogenic mechanism.
• the addictive form is a dose-dependent liver injury caused by the toxicity of the drug itself or its metabolites, caused by acetaminophen, methotrexate, and the like.
• the allergic idiosyncratic form is a dose-independent liver injury caused by autoimmunity due to the acquisition of antigenicity of the drug itself or its metabolites, ticlopidine HCl, loxoprofen Na, phenytoin, carbamazepine, rifampicin, tervinafin HCl Caused by such.
• Metabolic idiosyncratic type is a duration-dependent hepatic disorder caused by an increase in hepatotoxic metabolites due to genetic predisposition such as metabolic enzymes, and is caused by diclofenac Na, isoniazid, acarbose and the like. It is important to predict the onset of DILI for each patient before drug administration and to be able to select an appropriate drug from a plurality of drugs, and establishment of an evaluation system for that purpose is required.
• Patent Document 1 states that "a drug-metabolizing enzyme is used in a medium containing a drug to be tested, with a membrane that does not allow the cells to permeate the immune cells derived from the subject but allows the drug and its metabolites to permeate.
• a method for evaluating allergic drug-induced hepatocytes is described, which includes a step of co-culturing with expressed hepatocytes and a step of analyzing immune cells after the step.
• Non-Patent Document 1 also describes a DILI evaluation system in which a liver cell line (HepG2) and a monocyte / macrophage cell line (THP-1) are co-cultured, which is similar to Patent Document 1.
• Hepatocyte reactivity between thyroid cell line (HepG2) and a monocyte / macrophage cell line (THP-1) and non-DILI drugs (rosiglitazone, levofloxacin, acetylsalicylic acid, fluconazole) and with or without pro-inflammatory factors such as LPS and TNF It is stated that there was a difference.
• An object of the present invention is to provide a drug toxicity evaluation platform (evaluation method and a kit for the evaluation method, etc.) that enables detailed analysis of the possibility of drug-induced damage to the liver and other organs (DILI, etc.). And.
• liver organoids When liver organoids are co-cultured with blood cells (for example, immunocompetent cells such as monocytes / macrophages) and used as a DILI evaluation system, the present inventors accurately reflect the possibility of developing DILI due to drugs. I found that. Specifically, when the liver organoid is prepared using cells derived from a DILI patient, toxicity to the hepatic organoid is observed even with the addition of a small amount of DILI drug (for example, ampicillin which is an antibacterial drug), and the drug It was found that the possibility of developing DILI due to the disease can be evaluated with high sensitivity.
• DILI drug for example, ampicillin which is an antibacterial drug
• DILI is observed even if the liver organoid is prepared using cells derived from DILI patients. It has also been found that such drugs may not be available and can be determined to be potential alternatives to DILI patients.
• a drug toxicity evaluation method comprising a step of adding a drug to a co-culture system of an organoid and a blood cell and a step of evaluating the toxicity of the drug to the organoid.
• Item 2 The drug toxicity evaluation method according to Item 1, wherein the blood cells are immunocompetent cells.
• the drug toxicity evaluation method according to Item 1 or 2 is performed on a plurality of organoid samples, and a substance serving as a biomarker is obtained by comparing the substance in the culture supernatant of the sample in which drug toxicity occurs and the sample in which drug toxicity does not occur.
• a method of estimating a biomarker including the step of estimating.
• Item 3. A drug screening method comprising the step of performing the drug toxicity evaluation method according to Item 1 or 2 for a plurality of drugs using organoids derived from a patient who has developed drug toxicity, and selecting a drug having a low drug toxicity effect.
• a drug toxicity evaluation kit containing organoids and blood cells.
• Item 5. The kit according to Item 5, further comprising a dead cell detection reagent.
• the toxicity to the patient's organ can be accurately discriminated for each drug, and a drug with lower toxicity can be selected, or the toxicity of the drug can be determined. It will be possible to find a mechanism of action (biomarkers, etc.) for alleviation and new drugs with lower toxicity than before.
• the reason why the drug toxicity evaluation method of the present invention using organoids together with blood cells is excellent is that, for example, the three-dimensional organ structure including vascular endothelial cells is closer to the living body, and the drug toxicity and blood cells. It is presumed that the interaction between cells can be reproduced with high accuracy and that the activity of metabolic enzymes important for evaluation is high from the factors of cell maturation caused by cell-cell interactions. Further, since the drug toxicity evaluation method of the present invention uses blood cells, preferably immunocompetent cells, it is difficult to use the conventional evaluation method, especially when such cells (immune system) are involved in drug toxicity. In addition, the reproducibility of toxicity in vitro can be improved, and toxicity can be reproduced more accurately (with a high S / N ratio).
• the drug toxicity evaluation method of the present invention includes at least the following steps, and may further include other steps if necessary.
• Step 1 drug addition step: A step of adding a drug to a co-culture system of organoids and blood cells.
• Step 2 toxicity evaluation step: A step of evaluating the toxicity of the drug to the organoid.
• organ / tissue organoids used in the present invention
• the "organoid” used in the present invention includes organ or tissue organoids (collectively referred to as “organ / tissue organoids” in the present specification), cancer organoids, and the like.
• the organoids include not only organoids such as organs, tissues, and cancers whose structures are in the mature stage, but also simple cell aggregates (spheroids).
• Organoids may be derived from humans, or may be derived from animals other than humans, such as mammals such as mice, rats, dogs, pigs, and monkeys. That is, the predetermined cells used for producing organoids (see the description below for details) may be cells derived from humans or cells derived from animals other than humans. From the viewpoint of detecting a drug that causes drug addiction, which is difficult to find in conventional animal experiments or human cell tests in the development of human medicine, it is preferable that the organoid is derived from human. Further, the predetermined cell used for producing the organoid may be a primary cultured cell or a subcultured cell (strained cell).
• the organoid is derived from a patient (human) who has developed drug toxicity.
• the predetermined cells used for producing organoids are, for example, cells (primary cultured cells) collected from a patient who has developed drug toxicity, iPS cells produced using the cells, or them. It is a subcultured cell (strained cell) of.
• an iPS cell line prepared using cells collected from a patient who has developed drug toxicity can be used to prepare an organoid, that is, an organoid, in order to standardize the drug toxicity evaluation method of the present invention. It is suitable for inducing differentiation into predetermined various cells which are raw materials for the above.
• Organ / tissue organoid is an artificially created organ or tissue-like tissue (three-dimensional structure). Organoids of various types such as liver, pancreas, kidney, heart, lung, spleen, esophagus, stomach, thyroid gland, parathyroid gland, thymus, gonad, brain, spinal cord, skin, inner ear, etc. are already known. (For example, https://www.nejm.org/doi/pdf/10.1056/NEJMra1806175, https://www.nature.com/articles/s41568-018-0007-6, http://www.amsbio.com (See /brochures/organoid-culture-handbook.pdf). The "organ / tissue organoid” also includes "organ buds” (for example, liver buds and pancreatic buds), which are early-stage structures leading to organs having a complicated structure.
• the method for producing an organ / tissue organoid (organ bud) is known, and the method for producing the organ / tissue organoid used in the present invention is not particularly limited.
• the method for producing an organ / tissue organoid (organ bud) the cells constituting the organ / tissue, the mesenchymal cells, and the vascular endothelial cells as described in WO2015 / 129822 are co-cultured. Liver buds can be produced.
• organ / tissue organoid (organ bud) to be used can be selected according to the purpose of the drug toxicity evaluation method.
• an organoid of the liver which is an organ that actually causes a disorder (hepatocellular injury, etc.)
• Organoids such as skin and inner ear, which are organs in which an allergic reaction is likely to occur in the case of liver damage, may be used.
• a typical example of the organ organoid in the present invention is “liver organoid”.
• Liver organoids are preferably “liver buds”.
• a method for producing a liver organoid (liver bud) is known, and as a preferable example, according to the method for producing an organ organoid (organ bud) described in WO2015 / 129822, endoderm cells, mesenchymal cells and mesenchymal cells in the liver and A method of co-culturing vascular endothelial cells can be mentioned.
• a “cancer organoid” is a cell aggregate composed of cancer cells and other cells, and reproduces a cancer microenvironment.
• a method for producing a cancer organoid is known. For example, as described in Japanese Patent Application Laid-Open No. 2018-110575, a cancer organoid is produced by co-culturing cancer cells, mesenchymal cells and vascular endothelial cells. Can be done.
• the cancer cell may be an existing cancer cell line or a primary cancer cell line established using a cancer tissue isolated from the primary tumor of human cancer.
• the type of cancer is not particularly limited, and may be, for example, liver cancer, kidney cancer, malignant brain cancer, pancreatic cancer, gastric cancer, lung cancer and the like.
• the symptoms of cancer are exacerbated by producing an unusual metabolite in, for example, a drug-administered cancer cell (cancerous tissue) according to a typical embodiment of the present invention. It is possible to evaluate drug toxicity in a manner that causes or adversely affects other organs, and to estimate biomarkers involved in such drug toxicity.
• the drug toxicity evaluation method of the present invention can be changed to a method for evaluating a drug from a viewpoint other than toxicity, for example, a "drug resistance evaluation method".
• Treatment resistance (drug resistance, radiation sensitivity, immunotherapy sensitivity, nutrition therapy sensitivity, etc.) of cancer includes cancer cells and various cells (tumor-related fibroblasts) existing around the cancer cells. It is said that the tumor microenvironment constructed by the interaction of mesenchymal cells such as vascular endothelial cells and inflammatory cells such as macrophages plays an important role. Therefore, a drug susceptibility evaluation method including a step of adding a drug to a co-culture system of a cancer organoid and a blood cell (immunocytosis cell or the like) and a step of evaluating the drug susceptibility of the cancer organoid to the drug is carried out. It is also possible.
• the "cells that make up an organ / tissue” used to make organoids include (I) parenchymal cells that make up the organ / tissue and (II) non-parenchymal cells that make up the organ / tissue. Will be done.
• (I) parenchymal cells and (II) nonparenchymal cells include cells having predetermined functionality as parenchymal cells or nonparenchymal cells, which are (i) differentiated, matured, or reached terminal differentiation, respectively. (Simply referred to herein as "differentiated cells"), and (ii) capable of differentiating into parenchymal or non-parenchymal cells, or destined for differentiation (committed), but undifferentiated. Includes cells that are at the stage of, or stem or progenitor cells, and that do not yet fully possess the prescribed functionality as parenchymal or non-parenchymal cells (referred to herein simply as “undifferentiated cells”). Will be done.
• the cells constituting the organ / tissue at least one type of cell selected from the group consisting of differentiated cells of parenchymal cells, undifferentiated cells of parenchymal cells, differentiated cells of non-parenchymal cells and undifferentiated cells of non-parenchymal cells is preferable.
• Cell / tissue organoids eg, organ buds
• parenchymal cells constituting organs and tissues include liver hepatocytes, pancreatic endocrine cells (eg, ⁇ cells, ⁇ cells, ⁇ cells, ⁇ cells, PP cells), pancreatic duct epithelial cells, and renal urine.
• pancreatic endocrine cells eg, ⁇ cells, ⁇ cells, ⁇ cells, ⁇ cells, PP cells
• pancreatic duct epithelial cells eg. ⁇ cells, ⁇ cells, ⁇ cells, ⁇ cells, PP cells
• renal urine eg, ⁇ cells, ⁇ cells, ⁇ cells, ⁇ cells, PP cells
• Capillary epithelial cells and glomerular epithelial cells alveolar epithelial cells of the lung, myocardial cells of the heart, epithelial cells of the intestinal tract, nerve cells and glial cells of the brain, nerve cells of the spinal cord and Schwan cells and the like.
• non-parenchymal cells constituting organs / tissues include sinus endothelial cells of the liver, hepatic stellate cells and cupper cells, pancreatic stellate cells of the pancreas and pancreatic microvascular endothelial cells, renal glomerular endothelial cells of the kidney, and the like.
• Pulmonary pulmonary artery endothelial cells and pulmonary fibroblasts include heart cardiac microvascular endothelial cells, aortic endothelial cells, coronary artery endothelial cells and cardiac fibroblasts, intestinal intestinal microvascular endothelial cells, brain brain microvascular endothelial cells, perivascular Examples include cutaneous cells, choroidal endothelium cells and cerebrovascular outer membrane fibroblasts.
• Undifferentiated organ cells capable of differentiating into parenchymal cells or non-parenchymal cells constituting organs / tissues include, for example, brain, spinal cord, adrenal medulla, epiderm, hair / nail / skin gland, sensory organ, peripheral nerve.
• Cells that can differentiate into ectodermal organs such as the crystalline body; cells that can differentiate into mesodermal organs such as kidney, urinary tract, heart, blood, gonads, adrenal cortex, muscles, skeleton, dermis, connective tissue, and lining; liver , Cells capable of differentiating into endoderm organs such as pancreas, intestinal tract, lung, thyroid, parathyroid, urinary tract; and the like.
• hepatocytes used to make liver organoids (hepatoblasts) are parenchymal cells of the liver, which are differentiated hepatocytes (differentiated hepatocytes) and the fate of differentiation into hepatocytes has been determined, but the liver is still present. It is a term that includes the concepts of both undifferentiated cells (undifferentiated hepatocytes), so-called hepatic progenitor cells (eg, endoblasts in the liver). Differentiated hepatocytes may be cells collected from a living body (isolated from the liver in the living body), or to pluripotent stem cells such as ES cells and iPS cells, hepatic progenitor cells, and other hepatocytes.
• the undifferentiated hepatocytes may be those collected from a living body, or may be those obtained by differentiating pluripotent stem cells such as ES cells and iPS cells, and other stem cells or progenitor cells. ..
• Cells that can differentiate into hepatocytes include, for example, K.Si-Taiyeb, et al. Hepatology, 51 (1): 297-305 (2010), T. Touboul, et al. Hepatology. 51 (5): 1754- It can be prepared according to 65. (2010).
• pluripotent stem cells such as ES cells and iPS cells, hepatic progenitor cells, and other cells capable of differentiating into liver cells into liver cells are known.
• iPS cells Hepatology, 2010 ; 51 (1): 297-305, Cell Rep. 2017; 21 (10): 2661-2670, etc.
• a cell population collected from a living body or a cell population prepared by inducing differentiation of ES cells, iPS cells, etc. (especially for the latter) has a high purity of differentiated hepatic cells or a high purity of undifferentiated hepatic cells.
• a cell population may be used, or a cell mixture containing differentiated hepatocytes and undifferentiated hepatocytes in any proportion may be used.
• Whether or not a cell is a differentiated hepatocyte is determined by mature hepatocyte markers such as asialoglycoprotein receptor 1 (ASGR1), immature hepatocyte marker (early hepatocyte marker) ⁇ -fetoprotein (AFP), and early liver differentiation marker. It can be determined by whether the expression of one or more of albumin (ALB), retinol-binding protein (RBP4), transtiletin (TTR), glucose-6-phosphatase (G6PC) and the like is positive.
• ARB retinol-binding protein
• TTR transtiletin
• G6PC glucose-6-phosphatase
• a cell is an undifferentiated hepatocyte depends on whether the expression of one or more cell markers such as HHEX, SOX2, HNF4 ⁇ , AFP, and ALB is positive (weakly positive for ALB). It can be determined.
• vascular endothelial cells used for the production of organoids (organoid / tissue organoids, cancer organoids, etc.) are hematopoietic vascular endothelial cells (HEC) and non-hemogenic vascular endothelial cells (non-hemogenic endothelial cell; non).
• HEC hematopoietic vascular endothelial cells
• non-hemogenic vascular endothelial cell non-hemogenic endothelial cell
• -A term that includes both concepts of HEC HEC is a vascular endothelial cell capable of producing hematopoietic stem cells (having hematopoietic ability), and is also called a blood cell-producing vascular endothelial cell.
• non-HEC is a vascular endothelial cell that does not have such hematopoietic capacity.
• the vascular endothelial cells include vascular endothelial cells (for example, microvessel endothelial cells: MVEC), hepatic sinusoidal endothelial cells (LSEC), and umbilical-venal endothelial cells (umbilical-) collected from the living body. It may be a cell population with high purity (veinendothelial cells: UVEC), etc.), or it may be obtained by differentiating cells having the ability to differentiate into pluripotent stem cells such as ES cells and iPS cells, and other vascular endothelial cells. It may be a highly pure cell population of the obtained vascular endothelial cells.
• MVEC microvessel endothelial cells
• LSEC hepatic sinusoidal endothelial cells
• umbilical-venal endothelial cells umbilical-venal endothelial cells collected from the living body. It may be a cell population with high purity (veinendothelial cells: UVEC
• Mesenchymal cells used for the production of organoids (organ / tissue organoids, cancer organoids, etc.) are connected tissue cells that exist mainly in the connective tissue derived from the mesenchymal lobe and form the supporting structure of cells that function in the tissue. Differentiated cells (differentiated mesenchymal cells) and cells whose differentiation fate to mesenchymal cells has been determined but have not yet differentiated into mesenchymal cells (undifferentiated mesenchymal cells), so-called A term that includes both concepts of mesenchymal stem cells.
• vascular endothelial cells are a type of cells that differentiate from undifferentiated mesenchymal cells, they are excluded from the definition of "mesenchymal cells” in the present specification.
• Whether a cell is an undifferentiated mesenchymal cell or a differentiated mesenchymal cell can be determined, for example, by Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, which are markers of undifferentiated mesenchymal cells. , CD271, Nestin and the like can be discriminated by whether or not one or more species are positive (positive for undifferentiated mesenchymal cells, negative for differentiated mesenchymal cells).
• Mesenchymal cells further express cell markers specific to a specific organ (tissue) according to the organ organoids of the present invention and the "cells constituting the organ / tissue" used in combination. It may be a thing. Examples of such cell markers include FOXF1, COL4A and ALCAM, which are cell markers of septum transversum mesenchyme (STM).
• STM septum transversum mesenchyme
• the "blood cells” used in the present invention include erythrocytes, leukocytes (monocytes and granulocytes) and platelets.
• Monocytes include lymphocytes and monocytes.
• Lymphocytes include NK cells, T cells ( ⁇ T cells, ⁇ T cells, CD8 + T cells, CD4 + T cells, tumor infiltrating T cells, memory T cells, naive T cells, NKT cells) and B cells. Will be done.
• "Granulocytes” include neutrophils, eosinophils and basophils.
• the blood cells in the present invention are preferably immunocompetent cells.
• Immunogens correspond to the above-mentioned “monocytes”, and include T cells and B cells responsible for acquired immunity, and neutrophils, NK cells, monocytes, macrophages, dendritic cells, etc. responsible for innate immunity. Included.
• T cells, neutrophils, NK cells, macrophages and the like are preferable as blood cells (immunogens) that are co-cultured with organoids in the drug addition step.
• Blood cells may be cells collected from living organisms (eg, mononuclear cells isolated from peripheral blood), pluripotent stem cells such as ES cells and iPS cells, and others. It may be a cell obtained by differentiating a cell having the ability to differentiate into a blood cell. Further, the blood cell may be a primary cultured cell or a subcultured cell (strained cell).
• Blood cell cells may be derived from humans, or may be derived from animals other than humans, such as mammals such as mice, rats, dogs, pigs, and monkeys. From the viewpoint of detecting a drug that causes drug addiction, which is difficult to find in conventional animal experiments or human cell tests in the development of human medicine, blood cell cells are preferably derived from humans.
• the animal species of blood cell cells is usually the same as the animal species of organoids (predetermined cells used for their production).
• blood cell cells are derived from a patient (human) who has developed drug toxicity. That is, in a preferred embodiment of the present invention, a predetermined cell used for producing an organoid is produced, for example, using a cell (primary cultured cell) collected from a patient who has developed drug toxicity or the cell. These are iPS cells or their subcultured cells (strained cells).
• the "drug” used in the present invention is not particularly limited as long as it is a drug applicable to organs, tissues, etc. (including cancerous ones) for which organoids are produced, and toxicity is evaluated. Various drugs can be used depending on the purpose of use.
• toxicity caused by drugs includes not only toxicity to organs such as the liver as illustrated below, but also “drug eruption” (particularly allergic drug eruption).
• Drug eruptions include toxic epidermal necrolysis, Stevens-Johnson syndrome, and virus-related drug-induced hypersensitivity syndrome.
• the drug toxicity in the present invention is not limited to liver toxicity, and may be toxicity to organs or tissues other than the liver, such as cardiotoxicity and blood / bone marrow toxicity.
• the drug toxicity evaluation method of the present invention can be carried out using organoids of organs or tissues at which drug toxicity may develop (or should be verified).
• the drug is a drug for which it should be evaluated whether it has hepatic toxicity to the administration subject.
• Liver damage due to liver toxicity is generally classified into hepatocellular injury type, cholestasis type and mixed type thereof, or fulminant hepatitis.
• the mechanism of hepatotoxicity is classified into addictive type and idiosyncratic type, and idiosyncratic type may be further classified into metabolic idiosyncratic type and allergic idiosyncratic type.
• drugs related to each mechanism of action For example, the FDA (Food and Drug Administration) "Drug Induced Liver Injury Rank (DILIrank) Dataset" contains drugs related to drug-induced liver injury. It is published with the severity (Severity Class).
• various drugs for which drug toxicity has been reported, or various drugs that are (candidates for) their alternatives can be used.
• it may be recognized that it is related to a plurality of mechanisms of action for example, both allergic idiosyncratic type and metabolic idiosyncratic type).
• the drug itself or its metabolites are hepatotoxic, and almost all subjects (humans) develop liver injury in a dose-dependent manner.
• the addictive drug is relatively easy to reproduce in animal experiments and the like, it can also be used as a drug in the drug toxicity evaluation method of the present invention.
• addictive drugs include acetaminophen (aspirin) and methotrexate.
• a drug having a possibility of developing an idiosyncratic drug-induced liver injury is relatively difficult to reproduce in a toxicant experiment or the like, and can be said to be preferable as a drug in the drug toxicity evaluation method of the present invention.
• the drug itself or its reactive intermediate metabolite becomes a hapten and binds to various constituents of hepatocytes to acquire antigenicity, which causes an allergic reaction. Onset 1 to 8 weeks after taking the drug.
• allergic idiosyncratic drugs examples include ticlopidine HCl, loxoprofen Na, phenytoin, carbamazepine, rifampicin, and terbinafine HCl.
• metabolic idiosyncratic type it is thought that it develops due to individual differences in metabolic enzyme activity in the liver, and it is considered that the drug develops for a long period of 1 week (especially after 8 weeks) to 1 year or more. It develops when taken.
• Metabolic idiosyncratic drugs include, for example, acarbose, amiodarone, isoniazid, itraconazole, oral contraceptives, zafillucast, diclofenac sodium, disulfiram, tamoxyphene, anabolic steroids, danthrolen sodium, tegafur / uracil, tervinazone hydrochloride, troglitazone Discontinuation), sodium valproate, hydralazine hydrochloride, fluconazole, flutamide, pemorin, labetalol hydrochloride.
• direct hepatotoxic type drugs include high doses of acetaminophen, niacin, aspirin (acetylsalicylic acid), cocaine, IV amiodarone, IV methotrexate, and drugs used in cancer chemotherapy.
• Examples of the "specific hepatotoxic” type drug include amoxicillin / clavulanic acid, cephalosporin, isoniazid, nitrofurantoin, minocycline, fluoroquinolones, macrolides, antibodies and the like.
• Examples of "indirect hepatotoxic” type drugs include antitumor agents, glucocorticoids, monoclonal antibodies (TNF, CD20, checkpoint protein, etc.) (anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-). L1 antibody, etc.)), protein kinase inhibitor, etc.
• chemotherapeutic agents include rifampicin, isoniazid (isoniazid hydrazide: INH), salazosulfapyridine (sulfasalazine), ofloxacin, levofloxacin, norfloxacin, ciprofloxacin hydrochloride, sulfamethoxazole / trimethoprim, glyceofrubin, etc.
• Hepatobiliary type, mixed type, biliary stagnation type, fulminant hepatitis and other liver disorders have been reported.
• antibacterial agents include cephem (cefothium, cefaclor, cefazoline sodium, cefmethazole sodium, cephalexin, etc.), carbapenem (imipenem-silastatin sodium, etc.), penicillin (piperacillin sodium, ampicillin, amoxicillin, sulbactam).
• drugs that can develop drug-induced liver damage include, for example: Antipyretic anti-inflammatory analgesic ... Diclofenac sodium, acetaminophen, loxoprofen sodium, acetylsalicylic acid, mefenamic acid, ibuprofen, indomethacin, pranoprofen, sulindac, etc.; Psychiatric and neurological drugs: phenytoin, carbamazepine, sodium valproate, chlorpromazine hydrochloride, haloperidol, dantrolene sodium, halothane, phenytoin (diphenylhydantoin), pemorin, etc.); Cardiovascular drugs (including anticoagulants): Apringin hydrochloride, azimarin, trapidil, nifedipine, nicardipine hydrochloride, methyldopa, amiodarone, ticlopidine hydrochloride,
• TdP Torsade de Pointes
• hERG channel inhibitory action it should be evaluated whether the drug has cardiotoxicity to the administration subject.
• Cardiac toxicity is known to manifest, for example, as Torsade de Pointes (TdP), a lethal arrhythmia that causes sudden cardiac death.
• TdP risk the possibility of TdP generation by a drug (TdP risk) is evaluated by the presence or absence of QT prolongation (prolongation of action potential duration) of the myocardium, which is the previous stage, and the presence or absence of hERG channel inhibitory action. was there.
• the drug toxicity evaluation method of the present invention for example, by whether or not the waveform of the extracellular potential generated when a drug is allowed to act on a cardiac organoid conforms to a predetermined criterion, or by another method. , It is possible to assess whether the drug has cardiotoxicity (TdP risk).
• Various drugs that may cause cardiotoxicity are known and can be used in the present invention.
• antitumor agents such as anthracycline, cyclophosphamide, 5-fluorouracil, taxane; monoclonal antibodies such as trastuzumab, bevacizumab, nivolumab; tyrosine kinase inhibitors such as sunitinib, nilotinib; antiretroviral agents such as didobudin; Anti-diabetic drugs such as lociglycazone are mentioned as drugs having cardiotoxicity.
• Drug-induced blood and myeloid toxicity include, for example, erythrocytopenia, leukocyte (granulocyte) hypoplasia, thrombocytopenia, coagulation abnormality, bone marrow overgrowth, leukemia, 3 strains (erythroid system, granulocyte system and megakaryocyte system). Examples include panhemocyte hypocytosis with hematopoietic insufficiency. Blood and bone marrow toxicity is caused by hematopoietic disorders in the hematopoietic organs (mainly bone marrow) and peripheral blood cell destruction.
• Hematopoietic disorders occur when drugs act on myeloid stem cells and hematopoietic progenitor cells to suppress differentiation and proliferation. Blood cell destruction is often due to an immunological mechanism and is classified into drug adsorption type, immune complex type and autoimmune type.
• Various drugs that may cause blood / bone marrow toxicity are known and can be used in the present invention. Irinotecan, methotrexate, etc. are typical drugs that cause bone marrow (and associated blood cell) toxicity.
• antipyretic analgesics non-steroidal anti-inflammatory agents
• antipyretic analgesics non-steroidal anti-inflammatory agents
• antipsychiatric drugs anti-depressants
• chlorpromazine levomepromazine, chlordiazepoxide, meprobamate, clozapine
• antithyroid hormone drugs such as methylthiouracil, propylthiouracil, thiamazole
• diuretics such as chlortalidone, chlorothiazide, etaclinic acid
• Anticonvulsants antiepileptic drugs
• oral hypoglycemic agents such as chlorpropamide, tolbutamide
• the "drug” is not limited to the small molecule drug as exemplified above, and may be a drug other than various small molecule drugs that can be used as an active ingredient of the drug, such as an antibody drug, a peptide drug, and a nucleic acid drug.
• “Drugs” are not limited to drugs that are actually marketed as pharmaceuticals, but are drugs used in clinical trials or non-clinical studies, and drugs under development in the previous stage (candidate compounds for active ingredients of pharmaceuticals, etc.) ) May be.
• the drug addition step which is the first step in the drug toxicity evaluation method of the present invention, is a step of adding a drug to the co-culture system of organoids and blood cells.
• an appropriate medium may be selected according to the type of organoid.
• Mediums for organoids are known, and generally, a medium mixed with a medium used for culturing each of a predetermined cell for producing an organoid, for example, a medium for cells constituting an organ / tissue, a mesophyll.
• a mixed medium of a system cell medium and a vascular endothelial cell medium (the medium may be common) can be used as an organoid medium.
• the drug can be added to the above medium for organoids at a desired concentration suitable for evaluating the toxicity of the drug.
• the drug addition concentration is derived from the co-culture system (types and ratios of organoids, blood cells and drugs, medium composition, etc.), the evaluation method used in the toxicity evaluation process, or the cells used to prepare the organoids. It can be appropriately adjusted according to the attributes of the patient (for example, genes related to drug toxicity).
• the amount of the drug added in the evaluation method using a conventional culture system for various cells, organoids, etc. the drug in the evaluation method using the "co-culture system of organoids and blood cells" in the present invention.
• ampicillin should be 20 mg / mL or less (eg, concentrations at several points in the range 0-20 mg / mL, as well as numerical ranges for other drugs below).
• Amoxicillin can be added to the liver organoid medium at a concentration of 1.0 mg / mL or less, cephalexin at 2.0 mg / mL or less, and levofloxacin at 4.0 mg / mL or less.
• the toxicity evaluation step which is the second step in the drug toxicity evaluation method of the present invention, is a step of evaluating the toxicity of a drug to organoids.
• the type of drug toxicity refers to, for example, hepatocellular injury type, mixed type, cholestasis type, fulminant hepatitis, etc. in the case of liver toxicity.
• dead cells are detected by using a reagent (for example, propidium iodide; PI) among the cells that have undergone the drug addition step, and the ratio of dead cells in the cells (cell death rate). ), And the presence or absence of drug toxicity and the strength of drug toxicity can be evaluated by comparing with the control. It can be said that cell death due to the drug addition process reflects hepatocellular injury-type liver toxicity. For example, if the cell death rate is below a predetermined criterion, the drug tested can be evaluated as unlikely to have drug toxicity to the patient (low toxicity).
• a reagent for example, propidium iodide; PI
• the drug to be tested uses an organoid derived from a patient who develops drug toxicity, or when an organoid derived from a subject (healthy person) who has been confirmed not to develop drug toxicity is used.
• the above-mentioned cell death rate may be replaced with, for example, the "cell death increase rate" converted as a ratio to the cell death rate in the control performed without adding the drug to the medium.
• the drug toxicity evaluation method (drug addition step, toxicity evaluation step, etc.) of the present invention was performed on a plurality of organoid samples, and drug toxicity occurred (an index indicating drug toxicity is predetermined). Substances in the culture supernatant of specimens that were higher than the standard and were found to be drug toxic and those that did not occur (the index of drug toxicity was lower than the prescribed criteria and were not found to be drug toxic) Including a step of estimating a substance (eg, cytokine) that becomes a biomarker by comparing (eg, cytokine).
• a substance eg, cytokine
• the substance in the culture supernatant to be compared is a cytokine.
• Involvement of the immune system eg, release of inflammatory cytokines from T cells
• drug toxicity eg, liver damage.
• the drug toxicity evaluation method of the present invention it becomes possible to analyze the involvement of the immune system in drug toxicity and other causes.
• the culture supernatant of a sample whose cytotoxicity has been caused by a drug tested such as a patient-derived organoid
• cytokines inflammatory cytokines, etc.
• the concentration in the culture supernatant should be compared between a sample with cytotoxicity (organoids derived from patients, etc.) and a sample without cytotoxicity (organoids derived from healthy subjects, etc.). Therefore, the presence or absence of cytokines having a significantly higher concentration than the latter can be detected in the former sample.
• Cytokines with significantly higher concentrations in the culture supernatant of cytotoxic specimens can be presumed to be biomarkers of drug toxicity.
• Such a biomarker estimation method includes substances other than cytokines, for example, proteins other than cytokines, nucleic acid molecules such as miRNA, and nucleic acid molecules thereof, as long as they can be biomarkers that may be contained in the culture supernatant. It can also be applied to exosomes, metabolites (lipoxins derived from arachidonic acid, in particular, resolvins and protectins derived from ⁇ 3 fatty acids (DHA / EPA), etc.). causes other than the immune system may be involved in the mechanism of action of drug toxicity, and the substances to be compared in the culture supernatant can be selected according to the purpose of analysis.
• substances other than cytokines for example, proteins other than cytokines, nucleic acid molecules such as miRNA, and nucleic acid molecules thereof, as long as they can be biomarkers that may be contained in the culture supernatant. It can also be applied to exosomes, metabolites (lipoxins derived from arachid
• the drug toxicity evaluation method (drug addition step, toxicity evaluation step, etc.) of the present invention is carried out for a plurality of drugs using an organoid derived from a patient who has developed drug toxicity, and the drug toxicity effect is obtained. Includes the step of selecting a drug with a low value.
• drug with low drug toxicity effect means that the toxicity (indicator indicating) is less than or equal to a predetermined standard in the toxicity evaluation step in the drug toxicity evaluation method, or there is no significant difference from the subject. Refers to a drug that has been evaluated as having no drug toxicity.
• the drug screening method of the present invention determines, for example, a drug (which may be a commercially available drug or a drug in a test stage) that can be administered to a patient who develops drug toxicity. , It can be utilized to achieve both improvement of therapeutic effect and reduction of risk. For example, with respect to a drug (antibacterial drug, etc.) that may develop drug toxicity (liver toxicity, etc.), a plurality of candidate drugs to be administered to a patient from which the organoid is derived are selected, and the drug toxicity evaluation of the present invention is made for them. By implementing the method, it is possible to select a drug having a low drug toxic effect, that is, a drug that can be administered to the patient.
• a plurality of organoids derived from patients who have developed drug toxicity are prepared (for example, those having genes related to hepatotoxicity different from each other), and a drug having a low drug toxic effect for each organoid.
• the results of the selection can also be combined to select less toxic drugs that are less likely to cause cytotoxicity in patients of various attributes.
• the organoid may be an organoid derived from a subject whose drug toxicity is unknown, and a drug having a low drug toxic effect when administered to the subject is confirmed, and an appropriate drug is selected from among them. It is also possible to select it.
• kit of the present invention contains at least organoids and blood cells. Such a kit can be used to carry out the above-mentioned drug toxicity evaluation method of the present invention.
• the technical matters of the organoids and blood cells used in the kit are the same as those described in the present specification in relation to the drug toxicity evaluation method and the like.
• the kit of the present invention contains at least those derived from drug-toxic patients as organoids, and may further include those derived from healthy subjects (subjects who have not developed drug toxicity) as targets.
• the organoids derived from drug toxic patients may be one type or two or more types (for example, genes related to drug toxicity are different from each other).
• the kit of the present invention preferably further contains a dead cell detection reagent.
• the dead cell detection reagent include propidium iodide (PI).
• PI propidium iodide
• the kit in particular, depending on the evaluation method adopted in the toxicity evaluation step in the drug toxicity evaluation method of the present invention, the biomarker estimation method of the present invention or the method adopted in the drug screening method, cells.
• Other reagents may be used in place of or in addition to the detection reagents.
• the culture of the present invention comprises organoids, blood cells, and drugs.
• a culture corresponds to a culture prepared by the drug addition step in the drug toxicity evaluation method of the present invention and subjected to the toxicity evaluation step.
• the culture may be contained in a culture vessel such as a dish (Petri dish), or may be contained in a plurality of wells formed on a plate (contains in individual wells, or a medium thereof). It may be an aggregate of such inclusions).
• Example 1 Liver organoid toxicity test using an antibacterial agent [experimental method]
• the obtained mesodermal progenitor cells were further cultured in a medium containing Stempro-34 SFM (Gibco) (10 ml) supplemented with VEGF (200 ng / ml) and Folskolin (2 ⁇ M) at 5% CO 2 , 37 ° C. for 7 days.
• VEGF 200 ng / ml
• Folskolin 2 ⁇ M
• HE human liver endoblast cells
• the obtained endoderm progenitor cells were cultured in a medium containing 1% B27 Supplements (GIBCO) and FGF2 (10 ng / ml) in the basal medium at 5% CO 2 and 37 ° C. for an additional 5 days.
• GEBCO B27 Supplements
• FGF2 10 ng / ml
• MC human mesenchymal cells
• iPS cells derived from healthy humans (1383D2; iPS Research Institute, Kyoto University) and iPS cells derived from drug-induced liver injury patients (16-24; Yokohama City University)
• DMEM / F-12 Gibco
• GGIBCO B-27 Supplements
• BMP4 25 ng / ml
• CHIR99021 8 ⁇ M
• organoids three-dimensional structure
• HE human liver endoderm cells
• EC human vascular endothelial cells
• MC human mesenchymal cells
• Aggregates were prepared by mixing in (total 18 ⁇ 10 5 cells) and co-culturing in a three-dimensional culture vessel Elplasia (Kurare) for 1 day at 5% CO 2 and 37 ° C.
• the culture medium was HCM (Lonza) plus FBS (5%), HGF (10 ng / ml), OSM (20 ng / ml) and Dex (100 nM) for liver cells (A).
• Stempro-34 SFM (Gibco) mixed with VEGF (50 ng / ml) and FGF2 (10 ng / ml) medium for vascular endothelial cells (A) in a 1: 1 volume ratio (the present specification).
• Medium (referred to as "organoid medium") 2 ml was used.
• PBMC peripheral blood mononuclear cells
• PBMC was removed and the PI solution was was washed.
• a bright-field image and a fluorescence image (excitation wavelength: 530 nm, fluorescence wavelength: 620 nm) were obtained using a fluorescence microscope (KEYENCE BZ-X series, etc.).
• the rate of increase in dead cells was quantified by calculating the magnification of the PI staining rate with the drug not added as a control.

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