Drug Toxicity Evaluation Methods

Abstract

The present invention provides a drug toxicity evaluation platform (evaluation method and kit therefor, etc.) that makes it possible to perform a detailed analysis of the likelihood that a drug will cause injury (DILI, etc.) to the liver or another organ. This method for evaluating drug toxicity includes a step for adding a drug to a co-culture of an organoid and blood cells, and a step for evaluating the toxicity of said drug to said organoid.

Description

[Technical Field]

[0001] The present invention relates to an evaluation system that enables prediction of the possibility of drug-induced damage to the liver and other organs (for example, drug-induced liver injury). [Background technology]

[0002] Drug-induced liver injury (DILI) is a potential side effect of ongoing drug therapy. Drug-induced liver injury can be classified by mechanism into toxic, allergic, and metabolic idiosyncratic types. Toxic DILI is dose-dependent liver injury caused by the toxicity of the drug itself or its metabolites, and is induced by drugs such as acetaminophen and methotrexate. Idiosyncratic DILI is dose-independent liver injury caused by autoimmunity due to antigenicity of the drug itself or its metabolites, and is induced by drugs such as ticlopidine HCl, loxoprofen sodium, phenytoin, carbamazepine, rifampicin, and terbinafine HCl. Idiosyncratic DILI is duration-dependent liver injury caused by an increase in hepatotoxic metabolites due to genetic predisposition to metabolic enzymes, and is induced by drugs such as diclofenac sodium, isoniazid, and acarbose. It is important to predict the likelihood of DILI for each patient before administering a drug and to be able to select the appropriate drug from among several options, and there is a need to establish an evaluation system for this purpose.

[0003] Patent Document 1 describes a method for evaluating allergic drug-induced liver injury, which includes "a step of co-culturing immune cells derived from a subject with hepatocytes expressing a drug-metabolizing enzyme in a medium containing the drug to be tested, separated by a membrane that is impermeable to the cells but permeable to the drug and its metabolites," and "a step of analyzing the immune cells after the step."

[0004] Furthermore, Non-Patent Document 1 also describes an evaluation system for DILI similar to that described in Patent Document 1, in which a liver cell line (HepG2) and a monocyte / macrophage cell line (THP-1) are co-cultured, and describes that differences were observed in the reactivity of liver cells between DILI drugs (troglitazone, trovafloxacin, diclofenac, ketoconazole) and non-DILI drugs (rosiglitazone, levofloxacin, acetylsalicylic acid, fluconazole), and depending on the presence or absence of inflammatory factors such as LPS and TNF. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] JP 2016-202032 A (Patent No. 6516255) [Non-patent literature]

[0006] [Non-Patent Document 1] A. Granitzny et al. Toxicology Reports 4 (2017) 89-103 Summary of the Invention [Problem to be solved by the invention]

[0007] Although the evaluation systems for DILI listed in the Background Art have been proposed, there is still room for improvement in terms of enabling detailed analysis for each patient, such as being able to avoid drugs that may cause DILI and suggest alternative drugs.

[0008] An objective of the present invention is to provide a drug toxicity evaluation platform (e.g., an evaluation method and a kit therefor) that enables detailed analysis of the possibility of drug-induced damage to the liver and other organs (e.g., DILI). [Means for solving the problem]

[0009] The present inventors have found that when liver organoids are co-cultured with blood cells (e.g., immune cells such as monocytes / macrophages) and used as a DILI evaluation system, the likelihood of drug-induced DILI is accurately reflected. Specifically, when liver organoids are generated using cells derived from DILI patients, even the addition of small amounts of DILI drugs (e.g., the antibiotic ampicillin) is toxic to the liver organoids, enabling sensitive evaluation of the likelihood of drug-induced DILI. Furthermore, the addition of other drugs used for the same purpose as the above-mentioned DILI drugs (e.g., amoxicillin, cephalexin) sometimes does not result in DILI, even when liver organoids are generated using cells derived from DILI patients. It has also been found that such drugs can be identified as potential alternatives for DILI patients.

[0010] That is, in order to solve the above problems, the present invention provides the following [1] to [7]. [1] Adding a drug to a co-culture system of organoids and blood cells; and assessing the toxicity of the drug to the organoid. A method for evaluating drug toxicity, comprising: [2] Item 2. The drug toxicity evaluation method according to Item 1, wherein the blood cells are immunocompetent cells. [3] A method for estimating a biomarker, comprising the steps of: performing the drug toxicity evaluation method described in Item 1 or 2 on a plurality of organoid specimens; comparing substances in the culture supernatant of specimens in which drug toxicity occurred with those in which drug toxicity did not occur; and estimating substances that can serve as biomarkers. [4] A drug screening method comprising the steps of: performing the drug toxicity evaluation method according to item 1 or 2 for a plurality of drugs using organoids derived from patients who have developed drug toxicity, and selecting drugs with low drug toxicity effects. [5] A drug toxicity evaluation kit comprising an organoid and a blood cell. [6] Item 6. The kit of item 5, further comprising a dead cell detection reagent. [7] A culture comprising organoids, blood cells, and a drug. [Effects of the Invention]

[0011] By using the drug toxicity evaluation platform (e.g., evaluation method and kit therefor) according to the present invention, it becomes possible to accurately determine the toxicity of each drug to a patient's organs and select drugs with lower toxicity, as well as discover mechanisms of action (e.g., biomarkers) for mitigating drug toxicity and new drugs with lower toxicity than conventional drugs.

[0012] The drug toxicity evaluation method of the present invention, which uses organoids together with blood cells, is superior because, for example, the three-dimensional organ structure containing vascular endothelial cells is closer to that in vivo, and the interaction between drug toxicity and blood cells can be reproduced with high accuracy, and the activity of metabolic enzymes important for evaluation is high due to factors such as cell maturation caused by intercellular interactions. In addition, since the drug toxicity evaluation method of the present invention uses blood cells, preferably immunocompetent cells, it is possible to improve the reproducibility of in vitro toxicity, which was difficult with conventional evaluation methods, especially when such cells (immune system) are involved in drug toxicity, and to reproduce toxicity more accurately (with a high S / N ratio). DETAILED DESCRIPTION OF THE INVENTION

[0013] -Drug toxicity evaluation method- The drug toxicity evaluation method of the present invention includes at least the following steps, and may further include other steps as 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.

[0014] <Organoids> The term "organoid" as used herein includes organ or tissue organoids (collectively referred to herein as "organ / tissue organoids"), cancer organoids, etc. Organoids include not only organs, tissues, cancers, and other organoids that are at a mature stage, but also simple cell aggregates (spheroids).

[0015] Organoids may be derived from humans or mammals other than humans, such as mice, rats, dogs, pigs, monkeys, etc. That is, the predetermined cells used to prepare organoids (see below for details) may be cells derived from humans or cells derived from animals other than humans. From the viewpoint of detecting drugs that cause drug addiction that are difficult to discover in conventional animal experiments or human cell tests in the development of human medicines, organoids are preferably derived from humans. In addition, the predetermined cells used to prepare organoids may be primary cultured cells or subcultured cells (established cell lines).

[0016] In a preferred embodiment of the present invention, organoid is derived from a patient (human) who has developed drug toxicity.In this embodiment, the predetermined cells used to prepare organoid are, for example, cells (primary culture cells) collected from a patient who has developed drug toxicity, or iPS cells prepared using these cells, or their subcultured cells (established cell lines).For example, the iPS cell line prepared using cells collected from a patient who has developed drug toxicity is suitable for preparing organoid, that is, for inducing differentiation into various predetermined cells that will be the raw material for organoid, in order to standardize the drug toxicity evaluation method of the present invention.

[0017] "Organ and tissue organoids" are artificially created three-dimensional structures resembling organs or tissues. Various types of organoids, such as those of the liver, pancreas, kidney, heart, lung, spleen, esophagus, stomach, thyroid, parathyroid, thymus, gonads, brain, spinal cord, skin, and inner ear, are already known (see, for example, https: / / www.nejm.org / doi / pdf / 10.1056 / NEJMra1806175, https: / / www.nature.com / articles / s41568-018-0007-6, and http: / / www.amsbio.com / brochures / organoid-culture-handbook.pdf). "Organ and tissue organoids" also encompass "organ buds" (e.g., liver buds, pancreatic buds), which are early-stage structures that eventually develop into complex organs.

[0018] Methods for producing organ and tissue organoids (organ buds) are known, and the method by which the organ and tissue organoids used in the present invention are obtained is not particularly limited. A preferred example of a method for producing organ and tissue organoids (organ buds) is the method described in WO2015 / 129822, in which liver buds can be produced by co-culturing cells constituting organs and tissues, mesenchymal cells, and vascular endothelial cells.

[0019] The type of organ or tissue organoid (organ bud) to be used can be selected depending on the purpose of the drug toxicity evaluation method. For example, when performing a drug toxicity evaluation method for liver toxicity, liver organoids, which are the organ where damage (e.g., hepatocellular injury) actually occurs, can be used. Alternatively, organoids of the skin, inner ear, etc., which are organs prone to allergic reactions in cases of allergic-idiosyncratic drug-induced liver injury, can be used.

[0020] Representative example of organ organoid in the present invention is "liver organoid".Liver organoid is preferably "liver bud".Method for producing liver organoid (liver bud) is known, and a preferred example is the method of co-culturing liver endoderm cells, mesenchymal cells and vascular endothelial cells according to the method for producing organ organoid (organ bud) described in WO2015 / 129822.

[0021] "Cancer organoid" is a cell aggregate composed of cancer cells and other cells, which reproduces the cancer microenvironment. The method for producing cancer organoid is known, for example, as described in JP 2018-110575 A, cancer organoid can be produced by co-culturing cancer cells, mesenchymal cells and vascular endothelial cells.

[0022] The cancer cells may be an existing cancer cell line or a primary cancer cell line established using cancer tissue isolated from a primary human cancer focus. The type of cancer is not particularly limited, and may be, for example, liver cancer, kidney cancer, malignant brain tumor, pancreatic cancer, stomach cancer, lung cancer, etc.

[0023] In addition, when cancer organoids are used, a typical embodiment of the present invention can be used to evaluate drug toxicity, for example, by the production of unusual metabolites in drug-administered cancer cells (cancerous tissues), which can worsen cancer symptoms or adversely affect other organs, and further, to estimate biomarkers involved in such drug toxicity. On the other hand, when cancer organoids are used, the drug toxicity evaluation method of the present invention can also be modified to evaluate drugs from perspectives other than toxicity, such as a "method for evaluating drug resistance." It is believed that the tumor microenvironment established by the interaction between cancer cells and various cells surrounding the cancer cells (mesenchymal cells such as tumor-associated fibroblasts and vascular endothelial cells, inflammatory cells such as macrophages, etc.) plays an important role in the resistance to treatment (drug resistance, radiation sensitivity, immunotherapy sensitivity, nutritional therapy sensitivity, etc.) of cancers (e.g., pancreatic cancer). Therefore, a drug sensitivity evaluation method can also be performed, which includes the steps of adding a drug to a co-culture system of cancer organoids and blood cells (e.g., immunocompetent cells) and evaluating the drug sensitivity of the cancer organoids to the drug.

[0024] The "cells that make up organs and tissues" used to generate organoids (organ and tissue organoids) include (I) parenchymal cells that make up organs and tissues and (II) non-parenchymal cells that make up organs and tissues. Furthermore, (I) parenchymal cells and (II) non-parenchymal cells each include (i) differentiated, mature, or terminally differentiated cells that have the desired functionality of parenchymal or non-parenchymal cells (referred to herein simply as "differentiated cells"). Also included are (ii) cells that have the potential to differentiate into parenchymal or non-parenchymal cells or are committed to differentiation, but are undifferentiated or are at the stem or progenitor cell stage and do not yet fully possess the desired functionality of parenchymal or non-parenchymal cells (referred to herein simply as "undifferentiated cells").

[0025] The cells that make up organs and tissues can be at least one type of cell selected from the group consisting of differentiated parenchymal cells, undifferentiated parenchymal cells, differentiated non-parenchymal cells, and undifferentiated non-parenchymal cells, preferably a combination of two or more types of cells that can form "organ and tissue organoids" (e.g., organ buds).

[0026] Examples of "parenchymal cells" that make up organs and tissues include hepatocytes in the liver, endocrine cells (e.g., α cells, β cells, δ cells, ε cells, PP cells) and pancreatic duct epithelial cells, tubular epithelial cells and glomerular epithelial cells in the kidney, alveolar epithelial cells in the lung, cardiac myocytes, intestinal epithelial cells, neurons and glial cells in the brain, and neurons and Schwann cells in the spinal cord.

[0027] Examples of "non-parenchymal cells" that make up organs and tissues include sinusoidal endothelial cells, hepatic stellate cells, and Kupffer cells in the liver, pancreatic stellate cells and pancreatic microvascular endothelial cells in the pancreas, renal glomerular endothelial cells in the kidney, pulmonary artery endothelial cells and pulmonary fibroblasts in the lung, cardiac microvascular endothelial cells, aortic endothelial cells, coronary artery endothelial cells, and cardiac fibroblasts in the heart, intestinal microvascular endothelial cells in the intestine, cerebral microvascular endothelial cells, vascular pericytes, choroid plexus endothelial cells, and cerebrovascular adventitia fibroblasts in the brain.

[0028] Examples of "undifferentiated organ cells" that have the ability to differentiate into parenchymal or non-parenchymal cells that make up organs and tissues include cells that can differentiate into ectodermal organs such as the brain, spinal cord, adrenal medulla, epidermis, hair, nails, skin glands, sensory organs, peripheral nerves, and lens; cells that can differentiate into mesodermal organs such as the kidney, ureter, heart, blood, gonads, adrenal cortex, muscle, skeleton, dermis, connective tissue, and mesothelium; and cells that can differentiate into endodermal organs such as the liver, pancreas, intestine, lung, thyroid, parathyroid, and urinary tract.

[0029] The term "hepatocytes" used to produce liver organoids (liver buds) refers to liver parenchymal cells, and encompasses both differentiated hepatocytes (differentiated hepatocytes) and cells whose differentiation fate into hepatocytes has been determined but which have not yet differentiated into hepatocytes (undifferentiated hepatocytes), so-called hepatic progenitor cells (e.g., hepatic endoderm cells). Differentiated hepatocytes may be cells collected from a living organism (isolated from the liver in a living organism), or may be cells obtained by differentiating pluripotent stem cells such as ES cells and iPS cells, hepatic progenitor cells, or other cells capable of differentiating into hepatocytes. Undifferentiated hepatocytes may be collected from a living organism, or may be obtained by differentiating pluripotent stem cells such as ES cells and iPS cells, or other stem or progenitor cells. Cells that can be differentiated into hepatocytes can be prepared, for example, according to K. Si-Taiyeb, et al., Hepatology, 51 (1): 297-305 (2010) and T. Touboul, et al., Hepatology, 51 (5): 1754-65 (2010). Methods for differentiating pluripotent stem cells such as ES cells and iPS cells, hepatic progenitor cells, and other cells capable of differentiating into hepatocytes into hepatocytes are known. For example, iPS cells can be differentiated according to the methods described in Hepatology, 2010; 51 (1): 297-305, Cell Rep. 2017; 21 (10): 2661-2670, etc. For both cell populations collected from a living body and cell populations produced by inducing differentiation of ES cells, iPS cells, etc. (especially the latter), a cell population with a high purity of differentiated hepatocytes or a cell population with a high purity of undifferentiated hepatocytes may be used, or a cell mixture containing differentiated hepatocytes and undifferentiated hepatocytes in any ratio may be used.

[0030] Whether a cell is a differentiated hepatocyte can be determined by the positive expression of one or more of the following markers: mature hepatocyte markers, such as asialoglycoprotein receptor 1 (ASGR1), immature hepatocyte marker (early hepatic differentiation marker) alpha-fetoprotein (AFP), and early hepatic differentiation markers, such as albumin (ALB), retinol-binding protein (RBP4), transthyretin (TTR), and glucose-6-phosphatase (G6PC). On the other hand, whether a cell is an undifferentiated hepatocyte can be determined by the positive expression of one or more of the following cell markers: HHEX, SOX2, HNF4α, AFP, and ALB (weak positive expression for ALB).

[0031] The term "vascular endothelial cells," used to generate organoids (organ / tissue organoids, cancer organoids, etc.), encompasses both hemogenic endothelial cells (HECs) and non-hemogenic endothelial cells (non-HECs). HECs are endothelial cells capable of producing hematopoietic stem cells (having hematopoietic potential), also known as blood cell-producing endothelial cells. On the other hand, non-HECs are endothelial cells that lack such hematopoietic potential.

[0032] The vascular endothelial cells may be a highly purified cell population of vascular endothelial cells collected from a living body (e.g., microvessel endothelial cells (MVEC), liver sinusoidal endothelial cells (LSEC), umbilical-vein endothelial cells (UVEC)), etc.), or a highly purified cell population of vascular endothelial cells obtained by differentiating pluripotent stem cells such as ES cells and iPS cells, or other cells capable of differentiating into vascular endothelial cells.

[0033] "Mesenchymal cells," used to generate organoids (organ / tissue organoids, cancer organoids, etc.), refer to connective tissue cells that exist primarily in connective tissue derived from the mesoderm and form a support structure for cells that function in tissues. The term encompasses both differentiated cells (differentiated mesenchymal cells) and cells whose differentiation fate into mesenchymal cells has been determined but that have not yet differentiated into mesenchymal cells (undifferentiated mesenchymal cells), so-called mesenchymal stem cells. However, "vascular endothelial cells," a type of cell that differentiates from undifferentiated mesenchymal cells, are excluded from the definition of "mesenchymal cells" in this specification.

[0034] Whether a cell is an undifferentiated mesenchymal cell or a differentiated mesenchymal cell can be determined, for example, by whether it is positive for one or more of the markers for undifferentiated mesenchymal cells, such as Stro-1, CD29, CD44, CD73, CD90, CD105, CD133, CD271, and Nestin (if positive, it is an undifferentiated mesenchymal cell; if negative, it is a differentiated mesenchymal cell).

[0035] Mesenchymal cells may also express cell markers specific to a particular organ (tissue) depending on the organoid of interest or the cells constituting the organ or tissue used in combination with them. Examples of such cell markers include FOXF1, COL4A, and ALCAM, which are cell markers for septum transversum mesenchyme (STM).

[0036] <Blood cells> As used herein, "blood cells" include red blood cells, white blood cells (mononuclear cells and granulocytes), and platelets. "Mononuclear cells" include lymphocytes and monocytes. "Lymphocytes" include NK cells, T cells (αβT cells, γδT cells, CD8 + T cells, CD4 + "Granulocytes" include neutrophils, eosinophils, and basophils.

[0037] The blood cells in the present invention are preferably immunocompetent cells. "Immunocompetent cells" roughly corresponds to the above-mentioned "monocytes", and include the T cells and B cells that are responsible for adaptive immunity, and the neutrophils, NK cells, monocytes, macrophages, dendritic cells, etc. that are responsible for natural immunity.For example, T cells, neutrophils, NK cells, macrophages, etc. are preferred as the blood cells (immunocompetent cells) that are co-cultured with organoid in drug addition step.

[0038] Blood cells, preferably immunocompetent cells, may be cells collected from a living body (e.g., mononuclear cells isolated from peripheral blood), or may be cells obtained by differentiating pluripotent stem cells such as ES cells and iPS cells, or other cells capable of differentiating into blood cells. Furthermore, blood cells may be primary cultured cells or subcultured cells (established cell lines).

[0039] The blood cells may be derived from humans or from mammals other than humans, such as mice, rats, dogs, pigs, and monkeys. From the viewpoint of detecting drugs that cause drug addiction that are difficult to detect through conventional animal experiments or human cell tests in the development of human medicines, it is preferable that the blood cells are derived from humans. The animal species of the blood cells is usually the same as the animal species of the organoids (the specific cells used to produce them).

[0040] In a preferred embodiment of the present invention, blood cells are derived from the patient (human) who has developed drug toxicity.That is, in a preferred embodiment of the present invention, the predetermined cells used to prepare organoid are, for example, the cells (primary culture cells) collected from the patient who has developed drug toxicity or the iPS cells prepared by using these cells, or their subculture cells (established cell lines).

[0041] <Drugs> The "drug" used in the present invention is not particularly limited as long as it is a drug that can be applied to the organs, tissues, etc. (including cancerous ones) that are the target of organoid production, and various drugs can be used depending on the purpose of evaluating toxicity.

[0042] Drug toxicity includes not only toxicity to organs such as the liver, as exemplified below, but also drug rashes (especially allergic drug rashes). Drug rashes include toxic epidermal necrolysis, Stevens-Johnson syndrome, and even viral drug hypersensitivity syndromes.

[0043] Drug toxicity in the present invention is not limited to liver toxicity, but may also be toxicity to organs or tissues other than the liver, such as cardiac toxicity, blood or bone marrow toxicity, etc. The drug toxicity evaluation method of the present invention can be carried out using organoids of organs or tissues in which drug toxicity may occur (or whether this is possible should be verified).

[0044] In one embodiment of the present invention, the drug is a drug to be evaluated for its hepatotoxicity to a subject to be administered. Liver damage caused by hepatotoxicity is generally classified into hepatocellular injury, cholestatic injury, a mixed type thereof, or fulminant hepatitis.

[0045] The mechanisms of hepatotoxicity (drug-induced liver injury) are classified into toxic and idiosyncratic types, and the idiosyncratic type can be further classified into metabolic idiosyncratic and allergic idiosyncratic types. Numerous drugs have been reported to be involved in each mechanism of action. For example, the FDA's (Food and Drug Administration) "Drug Induced Liver Injury Rank (DILIrank) Dataset" publishes drugs involved in drug-induced liver injury along with their severity classes. The drug toxicity evaluation method of the present invention can also be used with various drugs for which drug toxicity has been reported, or various drugs that are substitutes (candidates) for these drugs. Note that the same drug may be recognized as being involved in multiple mechanisms of action (e.g., both allergic idiosyncratic and metabolic idiosyncratic).

[0046] Drugs that may cause toxic drug-induced liver injury are those in which the drug itself or its metabolites are hepatotoxic, causing dose-dependent liver injury in almost all subjects (humans). Toxic drugs are relatively easy to reproduce in animal experiments, and can also be used as drugs in the drug toxicity evaluation method of the present invention. Examples of toxic drugs include acetaminophen (aspirin) and methotrexate.

[0047] On the other hand, drugs that may cause idiosyncratic drug-induced liver injury are relatively difficult to reproduce in toxicology experiments, etc., and are therefore preferred as drugs for use in the drug toxicity evaluation method of the present invention. In the case of idiosyncratic allergic liver injury, it is believed that the drug itself or its reactive intermediate metabolites become haptens, which bind to various components of hepatocytes and acquire antigenicity, resulting in an allergic reaction. Most cases develop within 1 to 8 weeks after drug administration. Examples of drugs that cause idiosyncratic allergic liver injury include ticlopidine HCl, loxoprofen sodium, phenytoin, carbamazepine, rifampicin, and terbinafine HCl. In the case of idiosyncratic metabolic liver injury, it is believed that the onset is due to individual differences in metabolic enzyme activity in the liver, and develops after prolonged drug administration of one week (especially after 8 weeks) to one year or longer. Examples of drugs with metabolic idiosyncratic properties include acarbose, amiodarone, isoniazid, itraconazole, oral contraceptives, zafirlukast, diclofenac sodium, disulfiram, tamoxifen, anabolic steroids, dantrolene sodium, tegafur / uracil, terbinafine hydrochloride, troglitazone (discontinued), sodium valproate, hydralazine hydrochloride, fluconazole, flutamide, pemoline, and labetalol hydrochloride.

[0048] Recently, drug-induced liver injury has been classified into direct hepatotoxicity, idiosyncratic hepatotoxicity, and indirect hepatotoxicity (N Engl J Med 2019; 381:264-273). Examples of "direct hepatotoxic" drugs include high doses of acetaminophen, niacin, aspirin (acetylsalicylic acid), cocaine, IV amiodarone, IV methotrexate, and drugs used in cancer chemotherapy. Examples of "specific hepatotoxic" drugs include amoxicillin-clavulanate, cephalosporins, isoniazid, nitrofurantoin, minocycline, fluoroquinolones, macrolides, and antibodies. Examples of "indirect hepatotoxic" drugs include antitumor agents, glucocorticoids, monoclonal antibodies (anti-TNF, CD20, checkpoint proteins, etc. (anti-CTLA-4 antibody, anti-PD-1 antibody, anti-PD-L1 antibody, etc.)), and protein kinase inhibitors.

[0049] A more specific embodiment of the present invention is a method for evaluating liver toxicity caused by chemotherapeutic drugs (including antifungal agents) or antibacterial drugs. Chemotherapeutic drugs such as rifampicin, isoniazid (isonicotinic acid hydrazide: INH), salazosulfapyridine (sulfasalazine), ofloxacin, levofloxacin, norfloxacin, ciprofloxacin hydrochloride, sulfamethoxazole-trimethoprim, and griseofulvin have been reported to cause liver damage such as hepatocellular injury, mixed hepatitis, cholestatic hepatitis, and fulminant hepatitis. Antibacterial agents include, for example, cephems (cefotiam, cefaclor, cefazolin sodium, cefmetazole sodium, cephalexin, etc.), carbapenems (imipenem-cilastatin sodium, etc.), penicillins (piperacillin sodium, ampicillin, amoxicillin, sulbactam sodium-ampicillin sodium combination drug, oxypenicillin, clavulanate potassium-amoxicillin combination drug, tazobactam sodium-piperacillin sodium, Liver disorders such as hepatitis, mixed, cholestatic, and fulminant hepatitis have been reported with drugs such as cyclosporin (e.g., cyclosporin combination drugs), macrolides (e.g., erythromycin estolate, roxithromycin, clarithromycin, azithromycin), tetracyclines (e.g., tetracycline hydrochloride), minocycline hydrochloride, fosfomycin, cefteram pivoxil, cefpodoxime proxetil, flomoxef sodium, terbinafine hydrochloride, pyrazinamide, fluconazole, and itraconazole.

[0050] Drugs other than chemotherapy drugs and antibiotics that may cause drug-induced liver injury include, for example: Antipyretic, anti-inflammatory, analgesic drugs: 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), pemoline, etc.); Cardiovascular drugs (including anticoagulants): aprindine hydrochloride, ajmaline, trapidil, nifedipine, nicardipine hydrochloride, methyldopa, amiodarone, ticlopidine hydrochloride, hydrazine hydrochloride, labetalol hydrochloride, etc.; Gastrointestinal medications: tiopronin, famotidine, lansoprazole, cimetidine, sulpiride, omeprazole, ranitidine hydrochloride, etc.; Anticancer drugs: tegafur-uracil combination drug, cyclophosphamide, 6-mercaptopurine, cyclophosphamide, tamoxifen, flutamide, methotrexate, etc. Traditional Chinese medicines: Shosaikoto, Saireito, Kakkonto, etc. Agents for metabolic diseases (diabetes and hyperlipidemia): troglitazone, acarbose, voglibose, glibenclamide, epalrestat, etc.; Others: medications for gout and hyperuricemia, respiratory medications (e.g., zafirlukast), immunosuppressants (e.g., azathioprine), urinary and reproductive medications, bone metabolism improving medications, hormone medications (e.g., combination drugs of estrogen and progesterone preparations, anabolic steroids), antiallergic drugs, vitamin medications, over-the-counter medicines (e.g., vitamin A (retinol palmitate), acetaldehyde inhibitors (anti-alcohol therapy drugs, e.g., disulfiram), antithyroid drugs (e.g., propylthiouracil), etc.

[0051] In one embodiment of the present invention, a drug is evaluated for its cardiotoxicity to the subject to administration. Cardiotoxicity is known to manifest, for example, as Torsades de Pointes (TdP), a fatal arrhythmia that can cause sudden cardiac death. Previously, the possibility of a drug causing TdP (TdP risk) was assessed based on the presence or absence of myocardial QT prolongation (prolongation of action potential duration), a precursor to TdP, or the presence or absence of hERG channel inhibitory activity. In the drug toxicity evaluation method of the present invention, for example, when a drug is administered to cardiac organoids, the cardiotoxicity (TdP risk) of the drug can be assessed by determining whether the waveform of the extracellular potential generated meets a predetermined standard, or by other methods. A variety of drugs that may cause cardiotoxicity are known and can be used in the present invention. Examples of drugs that are cardiotoxic include antitumor drugs such as anthracyclines, cyclophosphamide, 5-fluorouracil, and taxanes; monoclonal antibodies such as trastuzumab, bevacizumab, and nivolumab; tyrosine kinase inhibitors such as sunitinib and nilotinib; antiretroviral drugs such as zidovudine; and antidiabetic drugs such as rosiglitazone.

[0052] In one embodiment of the present invention, drugs should be evaluated for their hematopoietic and bone marrow toxicity in the recipient. Examples of drug-induced hematopoietic and bone marrow toxicity include erythropenia, leukopenia (granulocytopenia), thrombocytopenia, coagulation disorders, abnormal bone marrow proliferation, leukemia, and pancytopenia accompanied by hematopoietic failure in three lineages (erythroid, granulocytic, and megakaryocytic). Hematopoietic and bone marrow toxicity are caused by hematopoietic disorders in the hematopoietic organs (mainly the bone marrow) and peripheral blood cell destruction. Hematopoietic disorders occur when drugs act on myeloid stem cells or hematopoietic progenitor cells, suppressing their differentiation and proliferation. Blood cell destruction is often due to immunological mechanisms and is classified into drug-adsorbed, immune complex, and autoimmune types. A variety of drugs with the potential to cause hematopoietic and bone marrow toxicity are known and can be used in the present invention. Irinotecan and methotrexate are representative drugs that cause bone marrow (and associated blood cell) toxicity. Drugs that cause granulocytopenia (drug-induced agranulocytosis) that leads to a decrease in neutrophils, etc., include, for example, antipyretic analgesics (non-steroidal anti-inflammatory drugs) such as aspirin, sulpyrine, diclofenac, indomethacin, etc.; antipsychotics (antidepressants) such as chlorpromazine, levomepromazine, chlordiazepoxide, meprobamate, clozapine, etc.; antithyroid hormone drugs such as methylthiouracil, propylthiouracil, thiamazole, etc.; diuretics such as chlorthalidone, chlorothiazide, ethacrynic acid, etc.; anticonvulsants (antiepileptic drugs) such as phenytoin, trimethadione, carbamazepine, etc.; These include oral hypoglycemic drugs such as chloramphenicol, penicillin, thiamphenicol, streptomycin, trimethoprim, and sulfamethoxazole; antibiotics and antibacterial drugs such as para-aminosalicylic acid (PAS) and isoniazid (INH); antihistamines such as cyproheptadine and chlorpheniramine; antirheumatic drugs such as phenylbutazone, indomethacin, and gold preparations; antiarrhythmic drugs such as procainamide, ajmaline, and quinidine; antiplatelet drugs such as ticlopidine; anti-ulcer drugs such as famotidine and lansoprazole; anticancer drugs, penicillamine, and sulfonamides (sulfa drugs).

[0053] The "drug" is not limited to the small molecule drugs exemplified above, but may be various drugs other than small molecule drugs that can be used as active ingredients in pharmaceuticals, such as antibody drugs, peptide drugs, and nucleic acid drugs.

[0054] The term "drug" is not limited to drugs that are actually commercially available as pharmaceuticals, but may also include drugs used in clinical trials or non-clinical trials, or drugs in the early stages of development (such as candidate compounds for the active ingredients of pharmaceuticals).

[0055] <Drug addition process> 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 a co-culture system of organoids and blood cells.

[0056] The culture medium for culturing organoids in the drug addition step can be selected from a suitable medium depending on the type of organoid. Culture media for organoids are known, and generally, a culture medium obtained by mixing media used to culture each of the specific cells for organoid production, such as a mixed medium for cells constituting organs and tissues, a medium for mesenchymal cells, and a medium for vascular endothelial cells (sometimes the same medium is used), can be used as the culture medium for organoids.

[0057] Drugs can be added to the organoid culture medium as described above at a desired concentration suitable for evaluating the toxicity of the drug. The concentration of the drug added can be appropriately adjusted according to the co-culture system (the type and ratio of organoids, blood cells, and drugs, the composition of the culture medium, etc.), the evaluation method used in the toxicity evaluation process, or the attributes of the patient from whom the cells used to prepare the organoids are derived (for example, genes related to drug toxicity). In addition, the amount of drug added in the evaluation method using the "co-culture system of organoids and blood cells" of the present invention can be adjusted by referring to the amount of drug added in the evaluation method using various conventional cell, organoid, etc. culture systems (for example, in consideration of the improvement of the evaluation accuracy according to the present invention, the numerical range (upper and lower limits) can be changed as necessary). For example, when evaluating liver toxicity using liver organoids, ampicillin can be added to the culture medium for liver organoids at concentrations of 20 mg / mL or less (e.g., several concentrations within the range of 0 to 20 mg / mL, the same applies to the numerical ranges of the other drugs below), amoxicillin at 1.0 mg / mL or less, cephalexin at 2.0 mg / mL or less, and levofloxacin at 4.0 mg / mL or less.

[0058] <Toxicity evaluation process> 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 an organoid.

[0059] The evaluation methods and standards in the toxicity evaluation process can be appropriately adopted depending on the types of organoids, blood cells, and drugs used, the type of drug toxicity, etc. For example, in the case of liver toxicity, the type of drug toxicity refers to hepatocellular damage, mixed type, cholestatic type, fulminant hepatitis, etc.

[0060] In one embodiment of the present invention, toxicity assessment involves detecting dead cells among cells that have undergone a drug addition step using a reagent (e.g., propidium iodide; PI), determining the proportion of dead cells among the cells (cell death rate), and comparing this with a control to assess the presence or absence of drug toxicity and the strength of drug toxicity. Cell death due to the drug addition step can be said to reflect hepatocellular damage-type liver toxicity. For example, if the cell death rate is below a predetermined standard, the tested drug can be evaluated as having a low likelihood of drug toxicity to patients (low toxicity). Alternatively, for example, the cell death rate results for a tested drug can be compared between organoids derived from patients who have developed drug toxicity and organoids derived from subjects (healthy individuals) confirmed not to develop drug toxicity. If there is no statistically significant difference, preferably if both cell death rates are below a predetermined standard, the tested drug can be evaluated as having a low likelihood of drug toxicity to patients (low toxicity). The above cell death rate may be replaced with the "cell death increase rate" calculated as a ratio relative to the cell death rate in a control where no drug was added to the medium, for example.

[0061] -Biomarker estimation method- The method for estimating biomarkers of the present invention includes a step of performing the drug toxicity evaluation method of the present invention (drug addition step, toxicity evaluation step, etc.) on multiple organoid samples, comparing substances (e.g., cytokines) in the culture supernatant of samples in which drug toxicity occurred (indicators representing drug toxicity are higher than a predetermined standard, and drug toxicity is recognized) with samples in which drug toxicity did not occur (indicators representing drug toxicity are lower than a predetermined standard, and drug toxicity is not recognized), and estimating substances (e.g., cytokines) that can serve as biomarkers.

[0062] In one embodiment of the present invention, the substance in the culture supernatant to be compared, i.e., the substance that serves as a biomarker, is a cytokine. The immune system (e.g., release of inflammatory cytokines from T cells) may be a contributing factor in the mechanism of drug toxicity (e.g., liver damage). However, it has not been elucidated whether the immune system is involved for all drugs, and there are drugs for which the immune system is not known to be involved but it is unclear whether it can truly be said that it is not involved. It is also possible that factors other than the immune system are involved in the mechanism of action. By applying 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.

[0063] For example, the culture supernatant of a sample in which a drug has caused cytotoxicity, such as a patient-derived organoid, is likely to contain a certain concentration of cytokines (e.g., inflammatory cytokines) released from the organoids. Therefore, by comparing the concentrations of various cytokines in the culture supernatant of a sample in which cytotoxicity has occurred (e.g., patient-derived organoids) with a sample in which cytotoxicity has not occurred (e.g., organoids derived from healthy individuals), it is possible to detect the presence or absence of cytokines in which the former sample has a significantly higher concentration than the latter. Cytokines with significantly higher concentrations in the culture supernatant of a sample in which cytotoxicity has occurred can be assumed to be biomarkers of drug toxicity.

[0064] This biomarker estimation method can also be applied to substances other than cytokines, as long as they are contained in the culture supernatant and can serve as biomarkers, such as proteins other than cytokines, nucleic acid molecules such as miRNA and exosomes containing them, and metabolic substances (lipid mediators, particularly arachidonic acid-derived lipoxins, and ω-3 fatty acid (DHA / EPA)-derived resolvins / protectins). Since factors other than the immune system may be involved in the mechanism of drug toxicity, the substances to be compared in the culture supernatant can be selected depending on the purpose of the analysis.

[0065] -Drug screening method- The drug screening method of the present invention includes a step of performing the drug toxicity evaluation method of the present invention (drug addition step, toxicity evaluation step, etc.) for multiple drugs using organoids derived from patients who have developed drug toxicity, and selecting drugs with low drug toxicity effects.

[0066] More specifically, a "drug with low drug toxicity effect" refers to a drug that is evaluated as having no drug toxicity, such as a toxicity (or an index representing toxicity) that is below a predetermined standard or that is not significantly different from the control, in the toxicity evaluation step of the drug toxicity evaluation method.

[0067] The drug screening method of the present invention can be used, for example, to determine which drugs (which may be commercially available pharmaceuticals or experimental drugs) can be administered to patients who have developed drug toxicity, and to achieve both improved therapeutic effects and reduced risk. For example, for drugs (antibacterial drugs, etc.) that may cause drug toxicity (liver toxicity, etc.), select multiple drugs that are candidates for administration to patients from whom organoids are derived, and then perform the drug toxicity evaluation method of the present invention on them, thereby selecting drugs with low drug toxicity effects, that is, drugs that can be administered to the patient.

[0068] Furthermore, the drug screening method of the present invention can also be used to select less toxic drugs that are less likely to cause cytotoxicity in patients with various attributes by preparing multiple organoids derived from patients who have developed drug toxicity (e.g., organoids with different genes related to liver toxicity), and integrating the results of selecting drugs with low drug toxicity effects for each organoid.

[0069] In addition, organoids may be derived from subjects whose risk of developing drug toxicity is unknown, and they can be used to identify drugs with low drug toxicity when administered to those subjects, and then select an appropriate drug from among them.

[0070] -kit- The kit of the present invention comprises at least organoid and blood cells.This kit can be used to carry out the drug toxicity evaluation method of the present invention described above.The technical matters of organoid 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.

[0071] The kit of the present invention comprises at least the organoid derived from drug toxicity patients, and can further comprise the organoid derived from healthy individuals (subjects that do not develop drug toxicity) as subjects.In addition, the organoid derived from drug toxicity patients can be one type, or two or more types (for example, the genes related to drug toxicity are different from each other).

[0072] The kit of the present invention preferably further comprises a dead cell detection reagent. Examples of the dead cell detection reagent include propidium iodide (PI). Depending on the use of the kit, particularly the evaluation method used in the toxicity evaluation step of the drug toxicity evaluation method of the present invention, or the method used in the biomarker estimation method or drug screening method of the present invention, other reagents can be used instead of or in addition to the cell detection reagent.

[0073] -Culture- The culture of the present invention comprises organoid, blood cell and drug.This culture corresponds to the culture that is prepared by drug addition process in the drug toxicity evaluation method of the present invention and is subjected to toxicity evaluation process.Culture can be contained in a culture vessel such as dish (petri dish), or can be contained in a plurality of wells formed on a plate (the contents in each well, or the collection of such contents). [Example]

[0074] [Example 1] Antibiotic toxicity test on liver organoids [Experimental Method]

[0075] (1) Preparation of human vascular endothelial cells (EC) Human iPS cells (1383D2; Kyoto University iPSC) derived from a healthy donor and iPS cells (16-24; Yokohama City University) derived from a patient with drug-induced liver injury were cultured in 10 ml of DMEM / F-12 (Gibco) supplemented with 1% B-27 Supplements (Gibco), BMP4 (25 ng / ml), and CHIR99021 (8 μM) at 37°C with 5% CO2 for 3 days. The resulting mesodermal progenitor cells were then cultured in 10 ml of Stempro-34 SFM (Gibco) supplemented with VEGF (200 ng / ml) and folskolin (2 μM) at 37°C with 5% CO2 for 7 days to generate CD31-, CD73-, and CD144-positive human vascular endothelial cell populations.

[0076] (2) Preparation of human hepatic endoderm (HE) cells Endodermal progenitor cells were induced from human iPS cells (1383D2; Kyoto University iPSC) and from a patient with drug-induced liver injury (16-24; Yokohama City University) by culturing them in 2 ml of basal medium (RPMI; Fujifilm) supplemented with Wnt3a (50 ng / ml) and activin A (100 ng / ml) at 5% CO2 and 37°C for 5 days. The resulting endodermal progenitor cells were then cultured for an additional 5 days at 37°C and 5% CO2 in the same basal medium supplemented with 1% B27 Supplements (GIBCO) and FGF2 (10 ng / ml). AFP-, ALB-, and HNF4α-positive human hepatic endoderm cell populations were obtained.

[0077] (3) Preparation of human mesenchymal cells (MCs) Mesodermal progenitor cells were induced from human iPS cells (1383D2; Kyoto University iPSC) and from a patient with drug-induced liver injury (16-24; Yokohama City University) by culturing them in 10 ml of basal mixed medium DMEM / F-12 (Gibco) supplemented with 1% B-27 Supplements (Gibco), BMP4 (25 ng / ml), and CHIR99021 (8 μM) at 37°C with 5% CO2 for 3 days. The resulting mesodermal progenitor cells were then cultured in the same medium supplemented with PDGFBB and activin A at 37°C with 5% CO2 for an additional 3 days. After culturing, the cells were collected and replated on new gelatin-coated plates. They were then cultured for an additional 4 days in the basal mixed medium supplemented with bFGF and BMP4 at 5% CO2 and 37°C to obtain a human mesenchymal cell population positive for FOXF1, COL4A, ALCAM, and CD73.

[0078] (4) Creation of organoids (three-dimensional structures) The prepared human hepatic endoderm cells (HE), human vascular endothelial cells (EC), and human mesenchymal cells (MC) were mixed at a cell number ratio of 10:7:1 (total number 18 × 10 5 The cells were mixed at 1000 x g (1000 x g) and co-cultured on a three-dimensional culture vessel (Elplasia, Kuraray) for 1 day at 5% CO2 and 37 °C to produce aggregates. In this co-culture, 2 ml of a 1:1 volumetric mixture of hepatic cell medium (A) (HCM, Lonza) supplemented with FBS (5%), HGF (10 ng / ml), OSM (20 ng / ml), and Dex (100 nM)) and a vascular endothelial cell medium (A) (Stempro-34 SFM, Gibco) supplemented with VEGF (50 ng / ml) and FGF2 (10 ng / ml) was used.

[0079] (5) Co-culture of organoids with peripheral blood mononuclear cells (PBMCs) and addition of antibiotics The prepared organoids were further cultured in organoid medium at 5% CO2 and 37°C for 114-15 days. After culture, the organoids were collected and seeded at 50-70 cells per well in a 96-well U-bottom low-attachment culture vessel (Corning, Sumitomo Bakelite, etc.). PBMCs (HEM) from multiple individuals, frozen and stored in a liquid nitrogen tank, were thawed and suspended in organoid medium supplemented with appropriate concentrations of antibiotics (ampicillin sodium, amoxicillin trihydrate, cephalexin, levofloxacin 0.5hydrate). The resulting PBMC suspension was then cultured at 2 x 10 cells per well. 5 PBMCs were added to the wells where the organoids had been seeded and mixed.

[0080] (6) Antimicrobial toxicity test using cell death detection reagent (propidium iodide) Organoids and PBMCs were co-cultured in antibiotic-containing medium at 5% CO2 and 37°C for 24 hours. Then, propidium iodide (PI, Dojindo Laboratories) was added to the medium and incubated at 5% CO2 and 37°C for 30 minutes. After incubation, 100 μL of culture supernatant was removed, and 100 μL of PBS(-) was added. The organoids were then allowed to settle for 1 minute, after which 100 μL of supernatant was removed. This process was repeated twice to remove the PBMCs and wash away the PI solution. After adding 100 μL of PBS(-), bright-field and fluorescent images (excitation wavelength: 530 nm, emission wavelength: 620 nm) were acquired using a fluorescence microscope (e.g., KEYENCE BZ-X series). The fluorescence intensity and organoid area of ​​the acquired images were quantified using the image analysis software Fiji, and the PI staining rate per organoid area was calculated (PI staining rate = PI fluorescence intensity / organoid area). The increase in cell death was quantified by calculating the fold increase in PI staining rate compared to the control without drug addition.

[0081] [result] (1) In this test system containing PBMCs, ampicillin significantly increased cell death in a concentration-dependent manner in liver organoids derived from the patient strain (16-24) that developed drug-induced liver injury (Table 1).

[0082] [Table 1]

[0083] (2) Toxicity tests were conducted using several antibiotics, and the compounds were classified into toxic compounds (ampicillin, levofloxacin 0.5 hydrate) and non-toxic compounds (amoxicillin trihydrate, cephalexin) (Table 2).

[0084] [Table 2]

Claims

1. Adding a drug to a co-culture system of liver organoids and blood cells; and Evaluating the hepatotoxicity of the drug on the liver organoid. Including; The liver organoid is derived from an iPS cell.

2. The method for evaluating liver toxicity according to claim 1 , wherein the blood cells are immunocompetent cells.

3. A method for estimating a biomarker, comprising the steps of: performing the liver toxicity evaluation method according to claim 1 or 2 on a plurality of liver organoid specimens; comparing substances in the culture supernatant of specimens in which liver toxicity has occurred with those in which liver toxicity has not occurred; and estimating substances that can serve as biomarkers.

4. A drug screening method comprising a step of performing the liver toxicity evaluation method according to claim 1 or 2 for a plurality of drugs using liver organoids derived from patients who have developed liver toxicity, and selecting drugs with low liver toxicity effects.

5. Use of a kit comprising liver organoids and blood cells in liver toxicity assessment, The liver organoid is derived from iPS cells.

6. The use according to claim 5, wherein the kit further comprises a dead cell detection reagent.

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