Pharmacokinetic evaluation kit and pharmacokinetic evaluation method using the same

Abstract

To provide a pharmacokinetic evaluation kit that enables study of the in-vivo pharmacokinetics of a drug in multiple organs by connecting multiple wells having organ cells arranged in the order of organs along the blood flow and / or in the order of organs related to the in-vivo pharmacokinetics of the drug to be measured, and a pharmacokinetic evaluation method using the kit.SOLUTION: A pharmacokinetic evaluation kit 1 includes a well plate 1a having a plurality of recessed wells 20 and 30 for accommodating test liquids 25, 35 and 26, 36 containing a drug to be measured for pharmacokinetics by organ-derived cells 22, 32 and 27, 37, the wells are interconnected, midway along the depth direction, by a channel 10a.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a pharmacokinetic evaluation kit and a pharmacokinetic evaluation method used to evaluate the pharmacokinetics of a drug that is orally administered to a living body, absorbed from the gastrointestinal tract, passes through the liver, metabolized in the liver, circulates systemically, is distributed to various organs, and is excreted in bile and urine; to evaluate the pharmacokinetics of a drug that is intravenously administered to a living body, passes through the blood vessels, circulates systemically, is distributed to various organs, is metabolized in the liver, and is excreted in bile and urine; and to evaluate the toxicity of a drug that is orally or intravenously administered to various organs when distributed to those organs. [Background technology]

[0002] Drugs administered to the body, for example, orally to a patient, are absorbed from the gastrointestinal tract through gastrointestinal cells and reach the liver via the portal vein. Some are metabolized and transported via the bloodstream to target organs where they exert their medicinal effects or produce side effects (toxicity). Some are excreted unchanged or as metabolites from the bile duct into the gastrointestinal tract along with bile. Others are excreted in urine or feces.

[0003] Drugs administered to the body and absorbed in the digestive tract undergo enterohepatic circulation or systemic circulation after being metabolized in the liver or without being metabolized in the liver. In particular, drugs metabolized by conjugation in the liver are excreted in the bile (biliary excretion), and then deconjugated by intestinal bacteria in the digestive tract to regenerate the unchanged drug, which can then be reabsorbed from the digestive tract (enterohepatic circulation).

[0004] For example, referring to Figure 7, which shows a schematic diagram of absorption, distribution, metabolism, and excretion (ADME), a portion of a drug that reaches the liver is excreted unchanged from the liver with bile and returns to the digestive tract via the bile duct (biliary excretion), where it is reabsorbed from the digestive tract, or a portion passes from the liver through the blood vessels to enter the systemic circulation and reach the target site (Figure 7(a)). Alternatively, the drug may be metabolized in the liver and excreted with bile, returning to the digestive tract via the bile duct. If metabolism in the liver is a conjugation reaction, the conjugated metabolites that return to the digestive tract are deconjugated and absorbed again via digestive cells (enterohepatic circulation). Alternatively, a portion passes from the liver through the blood vessels to enter the systemic circulation and reach the target site (Figure 7(b)). Alternatively, the drug may be metabolized in the liver and excreted in urine or bile.

[0005] During the development of new drugs and the evaluation of existing drugs, it is known that biliary excretion of the drugs or their metabolites plays an important role in the systemic clearance of the drugs.

[0006] However, in humans, the mechanisms of absorption, distribution, metabolism, and excretion are more complex. Furthermore, whether an administered drug is primarily excreted in the liver through bile or transferred to the bloodstream varies greatly depending on various factors, such as the physical properties of the drug, the species of the animal being administered, the susceptibility to conjugation metabolism such as glucuronidation, the contribution and extent of transporter activity, the susceptibility to micelle formation in the bile, and the extent of transfer into the bile.

[0007] Thus, even metabolism in the liver undergoes complex pharmacokinetics, but pharmacokinetics in multiple organs or throughout the body can be even more complex. Furthermore, pharmacokinetics can be toxic as drugs and their metabolites pass through multiple organs.

[0008] Traditionally, in vivo animal experiments have been used to assess whether biliary excretion or systemic distribution is dominant in humans. However, from the perspective of simple and easy pharmacokinetic studies and animal welfare, it is not appropriate to immediately conduct in vivo studies at the early stage of pharmacokinetic studies. Therefore, in recent years, methods based on inference from in vitro experiments have been developed to evaluate pharmacokinetics simply and easily.

[0009] For example, Patent Document 1 discloses a hepatocyte culture membrane in which at least any one of hepatic cancer cells, cholangiocarcinoma cells, free hepatocytes, animal-derived humanized fresh hepatocytes, iPS cells and / or ES cells induced to hepatocytes, organoids, and immortalized cells are cultured in a membranous cell layer on a porous plastic film, and the cells are oriented such that the lower side of the membranous cell layer faces the blood vessels and the upper side of the membranous cell layer faces the bile ducts. Patent Document 1 also discloses a drug transport evaluation kit comprising a hepatocyte culture membrane-supported insert container with a hepatocyte culture membrane at its bottom and containing a first test liquid, and a well plate with a well into which the hepatocyte culture membrane-supported insert container is inserted and containing a second test liquid in which the hepatocyte culture membrane is immersed.

[0010] The use of such a drug transport evaluation kit using hepatocyte culture membranes allows for the development of an orientation in which the lower side of the membranous cell layer, i.e., the porous plastic film side, faces the blood vessels and the upper side of the membranous cell layer faces the bile ducts. This makes it suitable for determining whether a target drug is preferentially excreted in bile or transported systemically. For example, when developing new drugs or evaluating existing drugs, it is possible to easily evaluate whether the target drug, such as the drug or its metabolite, is excreted in bile or pushed back into the blood vessels (sinusoids), thereby constructing a simple and highly reproducible evaluation system. Such a drug transport evaluation kit can be considered a test system closer to in vitro.

[0011] However, the pharmacokinetics of drugs usually involve the whole body, i.e., multiple organs, and undergoes the processes of absorption, distribution, metabolism, and excretion. During this process, pharmacokinetics is not determined solely by biliary excretion; as drugs circulate through organs via the blood, they exert their efficacy or side effects in the desired organs. Therefore, it is essential to examine the effects on various organs. Therefore, there has been a need for an improved, multi-organ pharmacokinetic evaluation kit that can examine the pharmacokinetics of drugs circulating through the blood between multiple organs, and that can serve as a test system that is closer to ex vivo than to in vivo or in vitro. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2021 / 132586 Brochure Summary of the Invention [Problem to be solved by the invention]

[0013] The present invention has been made to solve the above-mentioned problems, and aims to provide a pharmacokinetic evaluation kit that can be used to examine the pharmacokinetics of drugs in multiple organs by connecting multiple wells in which organ cells are placed in the order of the organs in blood circulation and / or the organs in which the drug is pharmacokinetically measured, and a pharmacokinetic evaluation method using the kit. [Means for solving the problem]

[0014] The pharmacokinetic evaluation kit developed to achieve the above-mentioned object is characterized by having a well plate in which a plurality of recessed wells for holding a test liquid containing a target drug whose pharmacokinetics is to be evaluated using organ-derived cells are connected by a passageway at the midpoint in the depth direction.

[0015] This pharmacokinetic evaluation kit preferably comprises the well plate in which the plurality of wells are connected by the passages to form a flow path for diffusing, distributing and / or circulating the test liquid.

[0016] This pharmacokinetic evaluation kit may comprise the well plate and a membranous cell culture membrane at the bottom, in which the organ-derived cells are arranged closely together on a porous plastic film in a single layer or in a mass or laminated state in which the organ-derived cells are closely overlapped, and an insert container that holds an organ-side test liquid and is inserted into and suspended in at least one of the plurality of wells to immerse the membranous cell culture membrane in the blood vessel-side test liquid, and the organ-derived cells may have a drug transport orientation such that the organ-derived cells are on the blood vessel side below the membranous cell culture membrane and on the organ side above the membranous cell culture membrane.

[0017] In this pharmacokinetic evaluation kit, the insert containers containing different organ-derived cells in the order of blood circulation organs and / or organs in the order of in vivo kinetics of the drug to be measured are inserted into and suspended in the multiple wells in sequence from upstream to downstream via the passage, and it is preferable that the wells are connected linearly, radially, or circularly via the passage.

[0018] This pharmacokinetic evaluation kit may have cultured cells in the form of a monolayer or non-monolayer membrane at the bottom of at least one of the wells on the upstream side.

[0019] In this pharmacokinetic evaluation kit, the flow path connecting the multiple wells may be parallel to the shortest distance line between the wells in the horizontal direction, or may be inclined at an angle of up to 45° in the horizontal direction from the shortest distance line.

[0020] In this pharmacokinetic evaluation kit, the well plate may have a water bath for heating to a constant temperature, or may be placed in a constant temperature bath.

[0021] In this pharmacokinetic evaluation kit, the multiple wells may have one or more depressions at the bottom of each well, each depression having a diameter of 100 μm to 1 mm and a depth of 20 μm to 40 μm, or the multiple wells themselves may be the depressions.

[0022] In this pharmacokinetic evaluation kit, the plurality of wells are preferably connected in a circular fashion, and at least one of the wells preferably has a circulator that circulates the blood vessel-side test liquid through the flow path.

[0023] In this pharmacokinetic evaluation kit, the circulatory organ is, for example, a stirrer bar.

[0024] In this pharmacokinetic evaluation kit, the stir bar preferably has a neodymium magnet.

[0025] In this pharmacokinetic evaluation kit, for example, the organ-derived cells are hepatocytes, gastrointestinal cells, bile duct epithelial cells or bile duct cells, renal tubular cells, cerebrovascular endothelial cells, vascular endothelial cells, or placental cells, which can be seeded in the form of a monolayer membrane, or hepatocytes, cardiac muscle cells, lung-derived cells, smooth muscle cells, or bile duct epithelial cells or bile duct cells, which can be seeded in the form of a mass or layer.

[0026] More specifically, this pharmacokinetic evaluation kit includes, as the organ-derived cells: the hepatocytes are at least any of hepatoma cells, free hepatocytes, fresh hepatocytes derived from animals or humans, iPS cells and / or ES cells induced to hepatocytes, spheroids and / or organoids of hepatocytes, and immortalized hepatocytes; the gastrointestinal cells are at least any one of gastrointestinal cancer cells, free gastrointestinal cells, fresh gastrointestinal cells derived from an animal or human, iPS cells and / or ES cells induced to become gastrointestinal cells, spheroids and / or organoids of gastrointestinal cells, and immortalized gastrointestinal cells; the bile duct epithelial cells or bile duct cells are at least any of cholangiocarcinoma cells, free bile duct epithelial cells, fresh bile duct epithelial cells derived from an animal or a human, iPS cells and / or ES cells induced to become bile duct epithelial cells or bile duct cells, spheroids and / or organoids of bile duct epithelial cells or bile duct cells, and immortalized bile duct epithelial cells or bile duct cells; the renal tubular cells are at least any of renal tubular cancer cells, free renal tubular cells, fresh renal tubular cells derived from an animal or human, iPS cells and / or ES cells induced to renal tubular cells, spheroids and / or organoids of renal tubular cells, and immortalized renal tubular cells; the cerebrovascular endothelial cells are at least any of cerebrovascular endothelial cancer cells, free cerebrovascular endothelial cells, fresh cerebrovascular endothelial cells derived from animals or humans, iPS cells and / or ES cells induced to cerebrovascular endothelial cells, spheroids and / or organoids of cerebrovascular endothelial cells, and immortalized cerebrovascular endothelial cells; the vascular endothelial cells are at least any one of vascular endothelial cancer cells, free vascular endothelial cells, fresh vascular endothelial cells derived from animals or humans, iPS cells and / or ES cells induced to become vascular endothelial cells, spheroids and / or organoids of vascular endothelial cells, and immortalized vascular endothelial cells; the placental cells are at least any of free placental cells, animal-derived humanized fresh placental cells, iPS cells and / or ES cells induced from placental cells, spheroids and / or organoids of placental cells, and immortalized placental cells; the cardiomyocytes are at least any of myocardial cancer cells, free cardiomyocytes, fresh cardiomyocytes derived from animals or humans, iPS cells and / or ES cells induced into cardiomyocytes, spheroids and / or organoids of cardiomyocytes, and immortalized cardiomyocytes; the lung-derived cells are at least any of lung-derived cancer cells, free lung-derived cells, fresh lung-derived cells derived from an animal or human, iPS cells and / or ES cells induced from lung-derived cells, spheroids and / or organoids of lung-derived cells, and immortalized lung-derived cells; The smooth muscle cells are at least any of smooth muscle-derived cancer cells, free smooth muscle-derived cells, fresh smooth muscle-derived cells derived from animals or humans, iPS cells and / or ES cells induced into smooth muscle-derived cells, spheroids and / or organoids of smooth muscle-derived cells, and immortalized smooth muscle-derived cells.

[0027] This pharmacokinetic evaluation kit may comprise an insert container having a monolayered membranous cell culture membrane made of the organ-derived cells on the porous plastic film at the bottom, and another insert container having a clumped or layered membranous cell culture membrane made of another organ-derived cell on another porous plastic film at the bottom, inserted into at least one of the multiple wells and suspended.

[0028] In this pharmacokinetic evaluation kit, for example, the cultured cells are hepatocytes, cardiac muscle cells, lung-derived cells, or smooth muscle cells that can be seeded in the non-monolayer form of a mass or laminated membrane.

[0029] Specifically, this pharmacokinetic evaluation kit includes the following cultured cells: the hepatocytes are at least any of hepatoma cells, free hepatocytes, fresh hepatocytes derived from animals or humans, iPS cells and / or ES cells induced to hepatocytes, spheroids and / or organoids of hepatocytes, and immortalized hepatocytes; the cardiomyocytes are at least any of myocardial cancer cells, free cardiomyocytes, animal-derived humanized fresh cardiomyocytes, iPS cells and / or ES cells induced into cardiomyocytes, cardiomyocyte spheroids and / or organoids, and immortalized cardiomyocytes; The lung-derived cells are at least any of lung-derived cancer cells, free lung-derived cells, fresh lung-derived cells derived from animals or humans, iPS cells and / or ES cells induced from lung-derived cells, spheroids and / or organoids of lung-derived cells, and immortalized lung-derived cells.

[0030] The pharmacokinetic evaluation method devised to achieve the above-mentioned object involves using the pharmacokinetic evaluation kit to culture cells derived from different organs in at least two or more of the multiple wells, adding a target drug to be measured, and measuring the concentration of the target drug in the test solution in the multiple wells via the passage, thereby evaluating pharmacokinetics.

[0031] In this pharmacokinetic evaluation method, it is preferable that the test liquid is diffused, distributed, and / or circulated within a flow path in which the plurality of wells are connected by the passage.

[0032] This pharmacokinetic evaluation method may involve providing a membranous cell culture membrane at the bottom of an insert container, in which the organ-derived cells are closely arranged on a porous plastic film in a single layer or in a clump or laminated state in which the organ-derived cells are closely overlapped, and then placing an organ-side test liquid therein and inserting the insert container into the multiple wells and suspending it, and immersing the membranous cell culture membrane in the vascular-side test liquid, which is the test liquid, so that the organ-derived cells have a drug transport orientation in which they are on the vascular side below the membranous cell culture membrane and on the organ side above the membranous cell culture membrane, and then adding the drug to be measured to the upstream well or the insert container, measuring the concentrations of the drug in the vascular-side test liquid in the well and the organ-side test liquid in the insert container, calculating the transport amount of the drug to be measured over time, and analyzing the transport rate to evaluate the pharmacokinetics. [Effects of the Invention]

[0033] The pharmacokinetic evaluation kit of the present invention is capable of handling the study of pharmacokinetics in multiple organs by having a well plate with multiple recessed wells, each containing a test solution containing a target drug whose kinetics are to be evaluated, connected by a passageway midway in the depth direction, while arranging organ cells in the order of the organs in blood circulation and / or the organs in which the target drug is kinetically distributed.

[0034] This pharmacokinetic evaluation kit can be used to examine pharmacokinetics in multiple organs in systemic circulation via blood, etc., when multiple wells are connected by passages to form a flow path for diffusing, distributing, and / or circulating the test liquid.

[0035] In this pharmacokinetic evaluation kit, if the flow path connecting multiple wells is parallel to the shortest distance line between the wells in the horizontal direction, or is inclined at a maximum of 45° from the shortest distance line in the horizontal direction, the flow within the cylindrical wells will be smooth, and the test liquid will be diffused, circulated, and / or circulated uniformly without stagnation.

[0036] In this pharmacokinetic evaluation kit, when a circulator, such as a stirrer bar with a neodymium magnet, is used to circulate the vascular side test liquid through a flow path in at least one of the multiple wells, there is almost no vibration, and there is no interference between multiple wells connected via a flow path, or between multiple wells in different series within the well plate that are not connected via a flow path, and the flow in the flow path is smooth and without unevenness.

[0037] This pharmacokinetic evaluation kit has a number of minute depressions in the bottom of at least one of its multiple wells, each having a diameter of several μm to 2 mm, preferably 10 μm to 1 mm, and more preferably 100 μm to 1 mm, and a depth of several μm to 1 mm, preferably 20 μm to 0.5 mm, and more preferably 20 μm to 40 μm.This allows even cells that have difficulty attaching and growing on a flat surface, such as hepatocytes, to accumulate in the depressions, where they can easily stick and attach, enabling them to be cultured in three dimensions.

[0038] This pharmacokinetic evaluation kit has an insert container that forms a membranous cell culture membrane with a membranous cell layer, formed by culturing various cells capable of forming various shapes, including hepatocyte-derived cells and hepatocyte-induced cells, in a monolayer, clump, or laminated form on a porous plastic film. The membranous cell culture membrane exhibits orientation, with the lower side, i.e., the porous plastic film side, facing the vascular side (blood side) and the upper side, i.e., the membranous cell layer side, facing the organ side. Because membranous cell layers, such as tightly packed monolayers or tightly packed clumps or laminates of hepatocyte culture membranes, exhibit orientation, they are suitable for investigating whether the target drug is preferentially transported to the organ side or the vascular side, such as biliary excretion or systemic uptake.

[0039] This pharmacokinetic evaluation kit has insert containers containing cells derived from different organs, each in the order of the blood circulation organs and / or the organs in which the drug to be measured is kinetically measured, inserted into multiple wells via passages from upstream to downstream, and connected in a linear, radial, or circular configuration.When this kit is used for developing new drugs or evaluating existing drugs, it is possible to easily evaluate whether the drugs to be measured, such as the drugs or their metabolites, are excreted in succession in various organs, pushed back into the blood vessels (sinusoids), or what absorption, distribution, metabolism, and excretion behavior they exhibit in multiple organs, or what toxicity and kinetics they exhibit.

[0040] When this pharmacokinetic evaluation kit has monolayer or non-monolayer cultured cells at the bottom of any of the wells, it becomes possible to examine not only the pharmacokinetics between the organ side and the vascular side, but also the pharmacokinetics on the vascular side.

[0041] This pharmacokinetic evaluation kit has multiple wells connected in a ring, and at least one of the wells has a circulatory system that circulates the vascular test liquid through a flow path, making it possible to reproduce the circulation of blood, etc., making it easy to accurately examine the pharmacokinetics of drugs in multiple organs in a short period of time.

[0042] This pharmacokinetic evaluation kit uses readily available cells, such as hepatocytes, hepatoma cells, cholangiocarcinoma cells, free hepatocytes, animal-derived humanized fresh hepatocytes, iPS cells and / or ES cells induced into hepatocytes, spheroids and / or organoids, and immortalized cells. Alternatively, it can be cultured in a non-monolayer fashion, such as gastrointestinal cells, bile duct epithelial cells or bile duct cells, renal tubular cells, cerebrovascular endothelial cells, vascular endothelial cells, placental cells, cardiac myocytes, lung-derived cells, or smooth muscle cells. By using these readily available cells, stable orientation can be achieved at low cost. The absorption, distribution, metabolism, and excretion behavior of these organ-derived cells can be reproduced in vitro. Therefore, pharmacokinetic evaluation kits using these organ-derived cells can be used for investigating the pathology of diseases and for drug discovery research into drug efficacy, toxicity, pharmacokinetics, etc. In particular, when the organ-derived cells are spheroids and / or organoids, especially organoids, they exhibit anatomical and functional characteristics similar to those of the organs they are derived from, making them useful for applied research and practical application in regenerative medicine using spheroids or organoids for transplantation.

[0043] This pharmacokinetic evaluation method using the pharmacokinetic evaluation kit can be used to easily and directly evaluate in vitro pharmacokinetics, such as the extent to which systemic circulation, in which the drug migrates from each organ to the blood vessels, and enterohepatic circulation, in which the drug migrates from the blood vessels to each organ, are predominant when the drug is administered to a human, as well as how the drug is absorbed, distributed, metabolized, and excreted in each organ, and whether toxicity occurs in each organ. Furthermore, this pharmacokinetic evaluation kit and pharmacokinetic evaluation method can reflect the function of each organ, allowing accurate indirect estimation of in vivo results without direct in vivo evaluation.

[0044] Furthermore, this pharmacokinetic evaluation kit and pharmacokinetic evaluation method can be used to easily evaluate pharmacokinetics by, for example, comparing the amount of the drug to be measured transported from the organ side to the blood vessel side with the amount of the drug to be measured transported from the blood vessel side to the organ side, or to evaluate differences in pharmacokinetics between organs.

[0045] Furthermore, this pharmacokinetic evaluation kit and pharmacokinetic evaluation method can easily, quickly, and accurately obtain ex vivo data that accurately reflect the physical properties of the drug being measured, particularly the pharmacokinetics such as toxicity and pharmacokinetics in humans, regarding the ease of systemic transfer from organs. [Brief explanation of the drawings]

[0046] [Figure 1] 1A and 1B are schematic cross-sectional and plan views showing an example of a two-well, non-circulating pharmacokinetic evaluation kit to which the present invention is applied. [Figure 2] FIG. 1 is a schematic plan view showing an example of a 5-well, non-circulating pharmacokinetic evaluation kit to which the present invention is applied. [Figure 3-1] FIG. 1 is a schematic plan view showing an example of a four-well type circulatory pharmacokinetic evaluation kit to which the present invention is applied. [Figure 3-2] FIG. 1 is a schematic plan view showing another example of a four-well type circulation-type pharmacokinetic evaluation kit to which the present invention is applied. [Figure 3-3a] FIG. 1 is a schematic plan view showing another example of a four-well type circulation-type pharmacokinetic evaluation kit to which the present invention is applied. [Figure 3-3b] FIG. 1 is a schematic plan view showing another example of a four-well type circulation-type pharmacokinetic evaluation kit to which the present invention is applied. [Figure 3-4] FIG. 1 is a schematic plan view showing another example of a 2-well to 4-well circulation-type pharmacokinetic evaluation kit to which the present invention is applied. [Figure 4] FIG. 1 is a schematic plan view showing an example of a six-well type circulatory pharmacokinetic evaluation kit to which the present invention is applied. [Figure 5]FIG. 1 is a schematic cross-sectional view showing an overview of pharmacokinetics in a circulating pharmacokinetics evaluation kit according to an embodiment of the present invention. [Figure 6] 1 is a graph showing the results of a drug transport evaluation method using a pharmacokinetic evaluation kit according to the present invention. [Figure 7] This is a diagram showing the absorption, distribution, metabolism, and excretion of a drug administered to the body and absorbed in the digestive tract, which then undergoes enterohepatic or systemic circulation without being conjugated or metabolized in the liver. DETAILED DESCRIPTION OF THE INVENTION

[0047] Hereinafter, the embodiments for carrying out the invention will be described in detail, but the scope of the present invention is not limited to these embodiments.

[0048] The pharmacokinetic evaluation kit and pharmacokinetic evaluation method of the present invention will be described in detail with reference to FIG. 1, focusing on a first embodiment of the simplest two-well type.

[0049] The pharmacokinetic evaluation kit 1 includes a well plate 1a having two recessed wells 20 and 30 arranged side by side, connected by a passage 10a at the midpoint of the depth of the recesses of the wells 20 and 30. In the impermeable plastic well plate 1a, the passage 10a and the wells 20 and 30 form a flow path so that the test solutions 26 and 36 contained in the wells 20 and 30 can diffuse, flow, and / or circulate via the passage 10a.

[0050] Insert containers 21 and 31 are inserted into at least one of the wells 20 and 30, and preferably into both wells, and are suspended by upper flanges 21a and 31a of the insert containers.

[0051] The insert container 21 has a bottom on which a membranous cell culture membrane 24 is provided, in the form of a single layer of organ-derived cells 22 closely arranged on a porous plastic film 23, or in the form of clumps or layers of cells closely overlapping each other. The membranous cell culture membrane 24 is immersed in a blood vessel-side test liquid 26 within the well 20 while containing an organ-side test liquid 25. For example, the organ-derived cells 22 may be gastrointestinal cells cultured in a monolayer, specifically, gastrointestinal cancer cells, free gastrointestinal cells, fresh gastrointestinal cells derived from animals or humans, iPS cells and / or ES cells induced into gastrointestinal cells, spheroids and / or organoids of gastrointestinal cells, or immortalized gastrointestinal cells. More specifically, one example of the organ-derived cells 22 is Caco-2 cells derived from human colon cancer. In this case, the drug transport orientation is such that the interior of the insert container 21, i.e., the upper side of the membranous cell culture membrane 24, is the organ side, i.e., the luminal side, and the exterior of the insert container 21, i.e., the lower side of the membranous cell culture membrane 24, is the blood vessel side.

[0052] A membranous cell culture membrane 24 is not particularly necessary when, for example, gastrointestinal cells have membrane permeability function and metabolic function, but if the metabolic function is weak or nonexistent depending on the type of gastrointestinal cells, gastrointestinal cells with metabolic function, such as free gastrointestinal cells or spheroids and / or organoids of gastrointestinal cells in a single layer or in an overlapping mass or layered form 27, may be attached to the bottom of the well 20, whereby metabolic function can also be examined.

[0053] When a test liquid containing a drug to be measured is placed in the insert container 21, the drug to be measured is absorbed through the membranous cell culture membrane 24, which is made of gastrointestinal cells, via the pathway B 20 As shown in the figure, the liquid moves from the organ-side test liquid 25 to the blood vessel-side test liquid 26, or along route A. 20 As shown in Fig. 1, the test fluid migrates again from the blood vessel side test fluid 26 to the organ side test fluid 25 and returns, and in some cases is affected by gastrointestinal cells having metabolic functions, such as free gastrointestinal cells, spheroids of gastrointestinal cells, and / or organoids in the form of a single layer or overlapping masses or layers of gastrointestinal cells 27, making it possible to examine gastrointestinal absorption, gastrointestinal metabolism, gastrointestinal toxicity, etc.

[0054] By measuring the concentrations of the drug to be measured in the organ-side test fluid 25 and the blood vessel-side test fluid 26 over time, the pharmacokinetics in the gastrointestinal cells can be investigated.

[0055] Meanwhile, the insert container 31 has a membranous cell culture membrane 34 at the bottom, in which organ-derived cells 32 are closely arranged on a porous plastic film 33 in the form of a single layer or in the form of a mass or laminate in which cells are closely overlapped, and the membranous cell culture membrane 34 is immersed in a blood vessel-side test liquid 36 within the well 30 while containing an organ-side test liquid 35. For example, the organ-derived cells 32 may be spheroids and / or organoids of bile duct epithelial cells cultured in a monolayer, manufactured by JSR Corporation. In this case, the inside of the insert container 31, i.e., the upper side of the membranous cell culture membrane 34, is the organ side, i.e., the bile side, and the outside of the insert container 31, i.e., the lower side of the membranous cell culture membrane 34, is the blood vessel side, resulting in a drug transport orientation.

[0056] A membranous cell culture membrane 34 is not particularly necessary when, for example, bile duct epithelial cells have membrane permeability and metabolic functions. However, when metabolic functions are weak or nonexistent, hepatocytes, specifically cells 37 in the form of a single layer or overlapping masses or layers such as human hepatoma-derived cell line HepG2 or well-differentiated human hepatoblastoma-derived cell line HuH-7, may be attached to the bottom of well 30 as needed, allowing metabolic functions such as liver metabolism and hepatotoxicity to be examined.

[0057] By measuring the concentrations of the drug to be measured in the organ-side test fluid 35 and the blood vessel-side test fluid 36 over time, it is possible to investigate, for example, the pharmacokinetics of the drug in gastrointestinal cells.

[0058] At this time, the target drug moves from the insert container 21 containing the test liquid containing the target drug to the organ-side test liquid 25 through the membranous cell culture membrane 24 to the blood vessel-side test liquid 26, and then diffuses, circulates, and / or circulates in the blood vessel-side test liquid 36 through the passage 10a. 30 As shown in the figure, the liquid moves from the blood vessel side test liquid 36 to the organ side test liquid 35 and back, or again along route B. 30As shown above, the substance moves from the organ-side test liquid 35 to the blood-side test liquid 36, and in some cases is affected by a single layer or overlapping masses or layers of hepatocytes 37, making it possible to study biliary excretion, hepatic metabolism, hepatotoxicity, etc.

[0059] Next, a second embodiment of the 5-well type pharmacokinetic evaluation kit 1 will be described in detail with reference to Figure 2. With regard to this pharmacokinetic evaluation kit 1, explanations of points that overlap with the first embodiment of the 2-well type will be omitted, and the explanation will focus on the differences between the 2-well type and the 5-well type.

[0060] The pharmacokinetic evaluation kit 1 in Figure 2 has the same internal configuration of well 30 as that in Figure 1, but compared to Figure 1, the central well 30 is connected to wells 20, 40, 50, and 60 on all four sides via passages 10a to 10d. In the pharmacokinetic evaluation kit 1 in Figure 2, insert container 21 is inserted into well 20 as in Figure 1, but no gastrointestinal cell device is present at the bottom of well 20. Well 30 is connected to wells 20, 40, 50, and 60 via passages 10b, 10c, and 10d, so that vascular test fluids 26 and 36 (see Figure 1) communicate with each of wells 40, 50, and 60. Well 40 has an insert container 41 inserted and suspended therein, but no cells are attached to the bottom. Well 50 does not use an insert container but has different cells attached to the bottom. Well 60 has an insert container inserted therein that is similar to wells 20 and 30 in Figure 1, but uses cells with a different membrane-like cell culture membrane.

[0061] An insert container 41 containing a membranous cell culture membrane (see FIG. 1; not shown in FIG. 2) consisting of a single layer of organ-derived cells 42, renal tubular cells, tightly arranged on a porous plastic film, is inserted into well 40 in the same manner as wells 20 and 30 in FIG. 1, and contains organ-side test liquid 45. To examine the urinary excretion and nephrotoxicity of the target drug, the upper side of the membranous cell culture membrane faces the organ, i.e., the urine side, and the lower side faces the blood vessel. Outside insert container 41 in well 40, the well communicates with blood vessel-side test liquid 36 (see FIG. 1) in well 30 via passage 10b.

[0062] To examine cardiotoxicity, clumps or layers of overlapping cardiomyocytes 52 are attached to the bottom of the well 50 as organ-derived cells 52. Within the well 50, the blood vessel side test liquid 56 communicates with the blood vessel side test liquid 36 (see FIG. 1) within the well 30 via the passage 10c.

[0063] An insert container 61 containing a membranous cell culture membrane (see FIG. 1; not shown in FIG. 2) consisting of a single layer of organ-derived cells 62, consisting of brain vascular endothelial cells tightly arranged on a porous plastic film, is inserted into well 60 in the same manner as wells 20 and 30 in FIG. 1, and contains organ-side test liquid 65. To examine the brain transfer and central toxicity of the target drug, the upper side of the cells on the membranous cell culture membrane faces the organ, i.e., the brain, and the lower side faces the blood vessels. Inside well 60, outside insert container 61, communication is established with blood vessel-side test liquid 36 (see FIG. 1) in well 30 via passage 10d.

[0064] When a test liquid containing a drug to be measured is placed in the insert container 21 in the well 20, the drug to be measured is absorbed through the membranous cell culture membrane 24, which is made of gastrointestinal cells, via pathway B. 20 As shown in the figure, the liquid moves from the organ-side test liquid 25 to the blood vessel-side test liquid 26, or along route A. 20 As shown in Fig. 1, the drug moves from the blood vessel-side test fluid 26 to the organ-side test fluid 25 and then back again. By measuring the concentrations of the target drug in the organ-side test fluid 25 and the blood vessel-side test fluid 26 over time, the pharmacokinetics of the drug in the gastrointestinal cells can be investigated.

[0065] The target drug migrates from the insert container 21 containing the test liquid containing the target drug through the membranous cell culture membrane 24 from the organ-side test liquid 25 to the blood vessel-side test liquid 26, and then diffuses, circulates, and / or circulates through the passage 10a into the blood vessel-side test liquid 36. The target drug that reaches the blood vessel-side test liquid 36 may migrate from the blood vessel-side test liquid 36 to the organ-side test liquid 35 through the membranous cell culture membrane 34, which is made of bile duct epithelial cells, and then back, or from the organ-side test liquid 35 to the blood vessel-side test liquid 36. In some cases, the target drug may be affected by hepatocytes 37, which may be in a single layer or in a clump or layer of overlapping cells (similar to Figure 1). By measuring the target drug concentrations in the organ-side test liquid 35 and the blood vessel-side test liquid 36 over time, the pharmacokinetics of the target drug in the hepatocytes can be investigated.

[0066] The vascular test solution 36 containing the target drug diffuses, flows, and / or circulates in wells 40·60. As in well 30, the target drug migrates from the vascular test solution in wells 40·60 to the organ test solution 45·65 and back via the membranous cell culture membranes of renal tubular cells (organ-derived cells 42) and the membranous cell culture membranes of cerebral vascular endothelial cells (organ-derived cells 62), or from the organ test solution 45·65 to the vascular test solution. By measuring the target drug concentrations in the organ test solution 45·65 and the vascular test solution over time for each well 40·60, the pharmacokinetics of the target drug in renal tubular cells and cerebral vascular endothelial cells can be investigated.

[0067] Furthermore, the vascular-side test liquid 36 containing the drug to be measured diffuses, flows, and / or circulates in the well 50. Within the well 50, the drug to be measured in the vascular-side test liquid 56 is affected by myocardial cells, which are organ-derived cells 52. By measuring the concentration of the drug to be measured in the vascular-side test liquid 56 over time, cardiotoxicity caused by myocardial cells can be investigated.

[0068] Next, a third embodiment of the active circulation 4-well type pharmacokinetic evaluation kit 1 will be described in detail with reference to Figure 3-1. In the pharmacokinetic evaluation kit 1 of Figures 1 and 2, even if the vascular test fluid diffuses, circulates, and / or circulates in each of the wells 20 to 60, this is limited to spontaneous diffusion, distribution, and / or circulation, which requires a considerable amount of time. In contrast, the pharmacokinetic evaluation kit 1 of Figure 3-1 is designed so that the vascular test fluid actively circulates in each of the wells 20 to 40, allowing for the investigation of the absorption, distribution, metabolism, and excretion of the drug as it circulates throughout the body. Regarding the pharmacokinetic evaluation kit 1 of Figure 3-1, explanations of the parts that overlap with the second embodiment of the 5-well type shown in Figure 2 will be omitted, and the following description will focus on the differences from the 5-well type.

[0069] The pharmacokinetic evaluation kit 1 in Figure 3-1 has the same internal configuration of wells 20, 30, and 40 as that in Figure 2, but compared to that in Figure 2, wells 20, 30, and 40 are connected in sequence via passages 10a and 10b, well 40 is connected to well 70 via passage 10c, and well 70 having a circulatory system 78 is connected to well 20 via passage 10e, making it an active circulation type.

[0070] In the pharmacokinetic evaluation kit 1, as shown in FIG. 3-1, the passages 10a, 10b, 10c, and 10e connect the adjacent cylindrical wells 20, 30, 40, and 70 along the shortest distance line 10a in the horizontal direction (for example, the horizontal plane at the midpoint of the depth of the wells 20, 30, 40, and 70). distance 10b distance 10c distance 10e distance As shown above, we have shown examples of connections, but as shown in Figure 3-2, the shortest distance line 10a distance 10b distance 10c distance 10e distance It is preferable that the wells 20, 30, 40, and 70 are connected so that they are parallel to the horizontal direction, for example, so that they are approximately tangent to each other, since this allows the water to flow uniformly within each well 20, 30, 40, and 70.

[0071] In the pharmacokinetic evaluation kit 1, the passages 10a, 10b, 10c, and 10e connect the adjacent cylindrical wells 20, 30, 40, and 70 along the shortest horizontal distance line 10a. distance 10b distance 10c distance 10e distance 3-3a, the passages 10a, 10b, 10c, and 10e may be tilted counterclockwise by θ (e.g., +30°), or clockwise by θ (e.g., -30°) as shown in FIG. 3-3b. θ is up to ±45°, but is preferably ±30° to 45°. When the passages 10a, 10b, 10c, and 10e are tilted by θ, preferably ±30° to 45°, the flow rate of the blood vessel-side test liquid (corresponding to 26, 36, etc. in FIG. 1) is stabilized, facilitating uniform diffusion, distribution, and / or circulation throughout the wells 20, 30, 40, and 70. This allows the pharmacokinetics of the target drug by the cells 22, 32, and 42 in the wells 20, 30, and 40 to be accurately and stably reflected.

[0072] The pharmacokinetic evaluation kit 1 is shown as an example having one set of four wells 20, 30, 40, and 70 in a well plate 1a as shown in Figures 1, 3-1 to 3-3a and 3-3b, but the kit may also have multiple sets of four wells 20, 30, 40, and 70 in the well plate 1a, for example, 2 sets, 4 sets, 6 sets, 8 sets, 12 sets, 16 sets, etc. (not shown).

[0073] As shown in Figures 3-4, which illustrate a 2-well, 4-well, or 6-well type set of pharmacokinetic evaluation kits 1, the well plates may be immersed in a water bath 110 that uses warm water 111 to maintain a constant temperature, with the warm water heated to 40-43°C, preferably about 40°C, so that the temperatures of the organ-side test fluid and the blood vessel-side test fluid are 36-37°C. Such well plates are heated by being placed on a spring net 112 stretched in the middle of the water bath. An aluminum plate 113 with a maximum thickness of 10 mm, preferably 5 mm, may be placed between the spring net 112 and the bottom of the well plate 1a to uniformly transfer heat to the bottom of the well plate 1a. Maintaining a constant temperature provides an environment suitable for the growth of cells 22, 32, and 42 within wells 20, 30, and 40, thereby enabling accurate and stable reflection of the pharmacokinetics of the target drug by cells 22, 32, and 42. Instead of being immersed in water bath 110, the pharmacokinetic evaluation kit 1 may be maintained at a constant temperature in a thermostatic bath, preferably heated to approximately 37°C, so that the temperature of the organ-side test fluid and the blood vessel-side test fluid is 36°C to 37°C (not shown).

[0074] The circulator 78 is used in conjunction with a magnetic stirrer (not shown) located below the well plate 1a so that the magnet rotates with a motor. It may be a magnetic stirrer bar with a built-in magnet that rotates in conjunction with the magnet and is coated with an inert resin such as polytetrafluoroethylene, or a rotor (not shown) located above the well plate 1a and rotated by a motor. The circulator 78 is preferably a magnetic stirrer bar used in conjunction with the magnetic stirrer, and may have a buffer or resistive material to prevent vibrations from being transmitted to the well plate 1a and inaccurately reflecting the pharmacokinetics of the target drug in the cells 22, 32, and 42 in each well 20, 30, and 40. For example, when the magnetic stirrer serving as the circulator 78 is rotated clockwise, a uniform concentration is quickly achieved, and the blood-side test fluid actively circulates through the wells 20, 30, 40, and 70, from the upstream side where it starts flowing from the well 70 to the downstream side where it returns to the well 70, just like blood. As a result, by placing cells 22, 32, and 42 derived from different organs in the order of blood circulation organs and / or organs in which the drug to be measured is kinetically transported in the insert containers 21, 31, and 41, respectively, from upstream to downstream, it becomes possible to examine the kinetics of the drug through multiple organs.

[0075] If the circulator 78 is a magnetic stirrer bar made of a ferrite magnet or a ferrite magnet coated with a tetrafluoroethylene film, the magnetic force is not very strong, so sufficient stirring may not be achieved unless the length is approximately 7 mm to 10 mm.In addition, it is difficult to adjust the rotation of the magnetic stirrer in any other way than in two stages, on and off, and the vibration is large, so it may interfere with the wells 20, 30, and 40 in the same group and / or the wells 20, 30, and 40 in other groups, making it difficult to ensure independence of the flow rate and the unevenness and uniformity of the flow. On the other hand, if the circulator 78 is a magnetic stirrer bar, and if it is made of neodymium or neodymium coated with a tetrafluoroethylene film, the magnetic force is strong, so even a short one, about 3 to 4 mm long, can provide sufficient stirring. In addition, the rotation speed can be easily adjusted with the magnetic stirrer, and since the stirrer bar is small, there is almost no vibration, so it is less likely to interfere with the wells 20, 30, and 40 in the same group and / or the wells 20, 30, and 40 in other groups, making it easier to ensure independence of speed and flow unevenness and uniformity.

[0076] The rotation speed of the magnetic stirrer bar is adjusted as appropriate, but it is preferable that the test liquid on the blood vessel side rotates once every 5 to 10 minutes.

[0077] Next, a fourth embodiment of the active circulation 6-well type pharmacokinetic evaluation kit 1 will be described in detail with reference to Figure 4. The pharmacokinetic evaluation kit 1 in Figure 4 actively circulates the vascular test fluid in each of the wells 20 to 60, and is designed to examine the absorption, distribution, metabolism, and excretion of the drug as it circulates throughout the body. Regarding the pharmacokinetic evaluation kit 1 in Figure 4, explanations of parts that overlap with the second embodiment of the 5-well type shown in Figure 2 and the third embodiment of the 4-well type shown in Figure 3-1 will be omitted, and the following description will focus on the differences from the 4-well type.

[0078] The pharmacokinetic evaluation kit 1 in Figure 4 has the same internal configuration of wells 20, 30, 40, 50, and 60 as that in Figure 2, but wells 20, 30, 40, 50, and 60 are sequentially connected via passages 10a, 10b, 10c, and 10d, well 60 is connected to well 70 via passage 10f, and well 70, which has a circulatory system 78, is connected to well 20 via passage 10g, making it an active circulation type. Similar to that in Figure 3-1, the pharmacokinetic evaluation kit 1 in Figure 4 has different organ-derived cells 22, 32, and 42 placed in insert containers 21, 31, and 41, respectively, in the order of blood circulation organs and / or organs involved in the pharmacokinetics of the drug to be measured, and by placing another organ-derived cell 52 in a well and a different organ-derived cell 62 in insert container 62 from upstream to downstream, it is possible to examine the pharmacokinetics through multiple organs.

[0079] In the pharmacokinetic evaluation kit 1 in Fig. 4, well 60 may be an empty well into which no insert container is inserted and air is bubbled into the vascular test liquid. Well 50 may contain an insert container provided with a membranous cell culture membrane (see Fig. 1, not shown in Fig. 2) having cardiomyocytes arranged in clumps or layers tightly on a porous plastic film.

[0080] 1-4 show examples of various cells placed in insert containers or wells. Specifically, gastrointestinal cells are used as the cells for the membranous cell culture membrane 24 in the insert container 21, and bile duct epithelial cells (organoids) are used as the cells for the membranous cell culture membrane 34 in the insert container 31, as shown in Figure 1. Other examples of cells for the membranous cell culture membrane include, for example, hepatoma cells, cholangiocarcinoma cells, free hepatocytes, animal-derived humanized fresh hepatocytes, iPS cells and / or ES cells induced into hepatocytes, spheroids and / or organoids, and / or immortalized cells. For example, clusters (aggregates) of various cells that are simply adhered to each other and have a three-dimensional structure but cannot self-renew are called spheroids, while precursor cells that can self-aggregate in the presence of a scaffold and differentiate to become liver-like cells through cell proliferation are called hepatic organoids.

[0081] Of these, hepatoma cells that are cultured to form membranous cell culture membranes 24-34 in insert containers 21-61 or to form non-monolayered, clumped, or laminated structures on the bottom of wells 20-60 are preferably commercially available hepatoma cell lines, such as the human hepatoma-derived cell line HepG2 (JCRB Cell Bank (National Institutes of Biomedical Innovation, Health and Nutrition)) and the well-differentiated human hepatoblastoma-derived cell line HuH-7 (JCRB, etc.). However, the human hepatoma-derived cell line HepaRG (KAC Co., Ltd.), PLC-PRF-5 (Cosmo Bio Co., Ltd., etc.), and the human liver adenoma cell line SK-HEP-1 (Cosmo Bio Co., Ltd., etc.) may also be used.

[0082] Examples of cholangiocarcinoma cells include commercially available liver cancer cell lines, such as the cholangiocarcinoma cell line TFK-1 (Medical Cell Resource Center, Institute of Development, Aging and Cancer, Tohoku University, National University Corporation), the human immortalized bile duct-derived cell line MMNK-1 (JCRB), the immortalized human hepatic endothelial cell line (JCRB), and sinusoidal endothelial carcinoma cells.

[0083] Examples of isolated hepatocytes include primary isolated hepatocytes, such as the human hepatocyte cell line Chang-Liver (Cosmo Bio Co., Ltd., etc.).

[0084] Examples of fresh animal-derived humanized hepatocytes include PXB-cells (PhoenixBio) and animal-derived hepatocytes in which human cells have been engrafted into immunodeficient animals (Central Institute for Experimental Animals, Public Interest Incorporated Foundation).

[0085] In the case of cultured precursor cells induced to become hepatocytes, examples of induced pluripotent stem cells (iPS cells) include iCell hepatocytes (Fujifilm Corporation) and Cellartis (Takara Bio Inc.).

[0086] Hepatocyte spheroids can be produced by shaking culture in non-adhesive culture plates (for example, non-adhesive surface cell culture, in which cells dropped onto a non-adhesive culture plate float in the medium and adhere to each other to form spheroids), adhesion methods that utilize surface tension (for example, hanging drop cell culture, in which suspended cells are dropped onto the inside of a culture plate lid and the droplets rise due to surface tension, and the cells gather downwards due to gravity and adhere to each other to form spheroids), and rotational culture methods (for example, rotary cell culture, in which the culture chamber is rotated to bring suspended cells into even contact with each other, producing spheroids with a relatively uniform cell number and size). Specifically, 3D InSight TM Liver Microtissues (InSphero) is an example.

[0087] In addition, hepatic organoids can be induced by culturing progenitor cells isolated from human livers with a specific combination of growth factors to form hepatic (or biliary) organoids. These can grow over the long term. For example, JSR Corporation's products can be used.

[0088] Immortalized hepatocytes include transformed cells.

[0089] Examples of hepatocytes have been given above as examples of cells that can be used for cells such as the membranous cell culture membranes 24-34 of the insert containers 21-61 and can be monolayered or non-monolayered, i.e., in a clumped or laminated state. In addition, other monolayered cells include the following:

[0090] For example, in order to examine the gastrointestinal absorption, metabolism, and toxicity of a drug to be measured, gastrointestinal cells that are formed into a monolayer such as a membranous cell culture membrane 24, 34 with the upper side facing the organ, i.e., the luminal side, and the lower side facing the blood vessels, include at least any of the following cells: gastrointestinal cancer cells such as Caco-2 cells derived from human colon cancer; free gastrointestinal cells that are derived from human or animal gastrointestinal cells and can be prepared in-house; iPS cells and / or ES cells induced into gastrointestinal cells, for example, manufactured by Fujifilm Corporation; spheroids and / or organoids of gastrointestinal cells, for example, manufactured by Medical Biofilm Research Institute Inc.; and immortalized cells derived from the gastrointestinal tract.

[0091] Examples of bile duct epithelial cells that can be used to make the upper side of the membranous cell culture membrane 24-34 the organ side, i.e., the luminal side, and the lower side the vascular side include cholangiocarcinoma cells derived from human cholangiocarcinoma cells, free bile duct epithelial cells that can be autologously prepared, for example, derived from human or animal bile duct epithelial cells, fresh animal-derived humanized bile duct epithelial cells, iPS cells and / or ES cells that can be induced into bile duct epithelial cells, spheroids and / or organoids of bile duct epithelial cells manufactured by JSR Corporation, and immortalized bile duct epithelial cells. For example, bile duct epithelial cell spheroids and / or organoids can be obtained by culturing bile duct epithelial cell spheroids and / or organoids in a growth factor-containing medium and Matrigel, and expanding them as needed. More specifically, bile duct cell spheroids and / or organoids can be seeded on a membranous cell culture membrane (e.g., Vitrigel®) in an insert container and cultured to form a cell monolayer, preferably with a ΔTEER of 100 Ω·cm. 2 Monolayers of the above can be formed, which can be cultured, passaged, and expanded long-term.

[0092] The upper side of the membranous cell culture membranes 24, 34, etc., is the organ side, i.e., the luminal side, and the lower side is the vascular side, and the urinary excretion and nephrotoxicity of the target drug are examined using renal tubular cells. Examples of such cells include tubular cancer cells such as LLC-PK1, which are porcine renal cancer cells (available from the RIKEN Cell Bank), and MDCK, which are canine renal cancer cells (available from American Type Culture Correction: ATCC), free renal tubular cells derived from animals or humans, fresh humanized renal tubular cells derived from animals, iPS cells and / or ES cells induced into renal tubular cells, spheroids and / or organoids of renal tubular cells, and immortalized renal tubular cells.

[0093] Examples of cerebrovascular endothelial cells for examining the brain transferability and central toxicity of a drug to be measured by placing the upper side of the membranous cell culture membrane 24-34 or the like on the organ side, i.e., the brain side, and the lower side on the blood vessel side include cerebrovascular endothelial cells that can be prepared in-house from bovine, rat, or mouse, iPS cells and / or ES cells induced to become cerebrovascular endothelial cells, spheroids and / or organoids of cerebrovascular endothelial cells, and immortalized human cerebrovascular endothelial cells such as HBMEC / ci18.

[0094] Examples of vascular endothelial cells for examining the vascular migration and vascular toxicity of a drug to be measured by placing the upper side of the membranous cell culture membrane 24·34, etc., on the organ side and the lower side on the vascular lumen side include vascular endothelial cancer cells, free vascular endothelial cells, fresh animal-derived humanized vascular endothelial cells, iPS cells and / or ES cells induced to vascular endothelial cells, spheroids and / or organoids of vascular endothelial cells, and immortalized vascular endothelial cells.

[0095] The placental cells used to examine the fetal transfer or fetal toxicity of a drug to be measured, with the upper side of the membranous cell culture membrane 24-34 or the like facing the organ side, i.e., the fetus side, and the lower side facing the blood vessel side, include at least any of the following: free placental cells that can be prepared in-house from bovine, rat, or mouse; fresh animal-derived humanized placental cells; iPS cells and / or ES cells induced into placental cells; spheroids and / or organoids of placental cells; and immortalized placental cells.

[0096] In addition to hepatocytes, examples of cells that are used to culture on the membranous cell culture membranes 24 / 34 of the insert containers 21 / 31 or on the bottom of the wells 20 / 30 and that form a non-monolayer, i.e., a clump or layer, include cardiac muscle cells, lung-derived cells, and smooth muscle cells.

[0097] Examples of cardiomyocytes used to examine the cardiomyocyte or cardiac delivery of a target drug, or its toxicity to cardiomyocytes or heart toxicity, include at least any of the following: cardiomyocyte cancer cells available from ATCC, free cardiomyocytes, fresh cardiomyocytes derived from animals or humans, iPS cells and / or ES cells induced into known cardiomyocytes, cardiomyocyte spheroids and / or organoids, and immortalized cardiomyocytes such as AC16 cells (Merck).

[0098] Lung-derived cells used to examine the migration or lung migration of a specific target drug into lung-derived cells or lung toxicity or lung toxicity include at least any of lung-derived cancer cells such as HCC827, A549, H441, and H1975 (available from ATCC), free lung-derived cells, fresh lung-derived cells derived from animals or humans, iPS cells and / or ES cells induced into lung-derived cells, spheroids and / or organoids of lung-derived cells, and immortalized lung-derived cells.

[0099] Examples of smooth muscle cells used to examine the smooth muscle delivery and smooth muscle toxicity of target drugs include smooth muscle cancer cells available from ATCC, free smooth muscle cells, fresh smooth muscle cells derived from animals or humans, iPS cells and / or ES cells induced into known smooth muscle cells, spheroids and / or organoids of smooth muscle cells, and immortalized smooth muscle cells.

[0100] Cells such as hepatocytes (parenchymal cells) used in these membranous cell culture membranes 24·34 etc. are seeded on porous plastic films 23·33 etc., preferably horizontally placed, and cultured in a medium, whereby the cells grow uniformly and tightly on the porous plastic films 23·33 etc. to form a monolayer of cultured cells that covers the porous plastic films 23·33 etc. In general, compared with endothelial cells, hepatocytes are less likely to form a membrane with orientation (polarity) like endothelial cells, even when cultured on porous plastic films, and a membranous cell culture membrane with excellent orientation has not been known. However, these cell culture membranes (24·34, etc.) are monolayer membranes in which cells such as gastrointestinal cells, hepatocytes, renal tubular cells, cerebral endothelial cells, and placental cells (22·32) are tightly packed together in a single layer. Each cell expresses vascular transporters on the porous plastic film (23·33, etc.) side, while expressing bile duct transporters on the opposite side. This allows specific drugs to be preferentially transported to the vascular or bile duct side. Furthermore, like the liver, they selectively transport specific drugs or glucose to the vascular side, while excreting certain drugs without allowing them to pass through. This allows them to function like the liver, with phase I (detoxification system), phase II (conjugation system), and phase III (excretion system). This results in an orientation where the lower side of the membrane-like cell culture membranes (e.g., 24·34) is adjacent to the blood vessels and the upper side of the membrane-like cell culture membranes (e.g., 24·34) is adjacent to the bile ducts. Drugs such as BSP (bromosulfophthalein, a biliary excretion marker), which is a substrate for the hepatic transporter MRP2 and enters the enterohepatic circulation at nearly 100% rate, are transported more to the bile ducts than to the blood vessels, demonstrating high orientation. When cultured cells (e.g., 22·32) are cultured on porous plastic films (e.g., 23·33), especially in bile acid-containing media, orientation becomes even more pronounced. This orientation, achieved by using membrane-like cell culture membranes (e.g., 24·34), can be used to investigate biliary excretion, hepatic metabolism, and hepatotoxicity.

[0101] The same applies to digestive tract cells, renal tubular cells, cerebrovascular endothelial cells, vascular lining cells, and placental cells other than hepatic cells that form a monolayer. In addition to hepatocytes, which form clumps or layers rather than monolayers, cardiomyocytes, lung-derived cells, and smooth muscle cells are also cultured in the same manner, except that they are cultured on the bottom of the well instead of on the porous plastic film of the insert container.

[0102] If such a plurality of wells have depressions, such as cylindrical depressions, with a diameter of about 100 μm to 1 mm and a maximum depression depth of 20 μm to 40 μm, cells can easily attach. Instead of depressions in the wells, such depressions themselves may serve as wells.

[0103] Referring to FIG. 1, an example of a membranous cell culture membrane 24 consisting of a layer of hepatocytes 22 and a porous plastic film 23 will be explained. The target drug to be measured, which is to be examined whether it is predominantly excreted in the bile from the liver or transferred to the whole body, is transported in the test solution from the blood vessel side B to the bile duct side A of the membranous cell culture membrane 24 (P BtoA ) and the transport rate from bile duct side A to blood vessel side B (P AtoB ) may vary depending on the drug to be measured, but is 1.3 or higher, preferably 1.8 or higher, more preferably 2.0 or higher, and even more preferably 5.0 or higher. This ratio is expressed by the following mathematical formula (1).

number

[0104] In addition to the above indexes, when calculating the ratio of transport rates when an inhibitor (inhibitory substance), such as a transporter inhibitor, is added, the transport rate from the vascular side B to the bile duct side A (P BtoA ) and the transport rate from bile duct side A to blood vessel side B (P AtoB ) is the transport rate from the vascular side B to the bile duct side A when an inhibitor is added (P BtoA i ) and the transport rate from bile duct side A to blood vessel side B (P AtoB i) may vary depending on the drug to be measured, but may be 1.3 or more, preferably 1.8 or more, more preferably 2.0 or more, and even more preferably 5.0 or more. This ratio is expressed by the following formula.

number

[0105] When the value of equation (1) is 1 or more and both orientations are observed, the ratio of transport rates (P BtoA i ) / (P AtoB i ) is calculated, the value becomes small and orientation disappears or decreases, indicating that the transporter is functioning in the direction that orients the movement.

[0106] On the other hand, if it is better to add an inhibitor and determine the transport rate ratio, use the (P BtoA ) / (P AtoBWhen the value of (2) is less than 1, the target drug is likely to be transported toward the bloodstream. Specifically, when an inhibitor is added and the transport rate is measured, if a large value of equation (2) is obtained, it appears that the target drug is transported toward the bloodstream, but biliary excretion is also functioning. In this case, an inhibitor that inhibits the transport of the target drug is required. More specifically, carboxy-2,7-dichlorofluorescein (CDCF) transports in HepG2 cells via excretory transporters expressed on the bile duct side, such as multidrug resistance-associated protein 2 (MRP2), P-glycoprotein (P-gp), and breast cancer resistance protein (BCRP), an ABC transporter (ATP-binding cassette transporter). Therefore, MK-571, which inhibits these transporters, is used as an inhibitor. As another example, in the case of P-glycoprotein (P-gp) instead of MRP2, rhodamine 123 (Rho123) is used instead of CDCF, and verapamil or cyclosporine is used as an inhibitor.

[0107] Even if these hepatocytes are cultured in ordinary petri dishes or plastic well plates, they exist randomly and not only do they not show orientation, but they also gradually form colonies or three-dimensional masses or multiple layers.

[0108] Therefore, to prepare membranous cell culture membranes such as this hepatocyte culture membrane 24·34, it is important to culture hepatocytes on porous plastic films 23·33.

[0109] Examples of materials for the porous plastic films 23 and 33 in the cell culture membranes 24 and 34 include fluorine-containing resins such as polytetrafluoroethylene (PTFE), polycarbonate, polyolefins such as polyethylene, polypropylene, and polystyrene, polyesters such as polyethylene terephthalate, polyurethane, polyamide, polyimide, cellulose, and regenerated cellulose. The porous plastic films 23 and 33 may be plastic films or nonwoven fabrics.

[0110] The pores of the porous plastic films 23-33, etc., are smaller than the size of an individual hepatocyte (approximately 10 μm), preventing hepatocytes from passing through, but are larger than the molecular diameter of the target drug molecules or hydrates thereof in the test solution, allowing them to pass through. Specifically, the pore size of the pores is, for example, 0.1 μm to 10 μm, preferably 0.3 μm to 8 μm, and more preferably, the average pore size of the porous plastic films 23-33 included in commercially available empty kits is 0.3 μm, 0.4 μm, 1.0 μm, 3.0 μm, 5 μm, or 8.0 μm. The thickness of the porous plastic films 23-33, etc., is, for example, 1 μm to 100 μm, preferably 10 μm to 30 μm.

[0111] Porous plastic films 23, 33, etc. may be tissue culture treated to optimize cell attachment and proliferation, and may be coated with collagen and / or fibronectin. Porous plastic films may also be patterned. Collagen-coated porous plastic films have improved cell adhesion due to the collagen.

[0112] The wells 20 to 70 are recessed, for example, into a plastic well plate 1a having 12 to 96 wells (12 holes), with a diameter of 5 mm to 30 mm, preferably 8 mm to 25 mm, and a depth of 10 mm to 20 mm, preferably 15 mm to 20 mm, and more preferably 17.5 mm, and are connected to each other by passages 10a to 10f, which are cylindrical with a diameter of 1 mm to 5 mm, preferably 3 mm to 4 mm, or rectangular prism-shaped with a side of 1 mm to 5 mm, preferably 3 mm to 4 mm.

[0113] For example, plastic well plates 1a are commonly arranged in 3 wells x 4 wells for 12 wells, 4 wells x 6 wells for 24 wells (24 holes), and 8 wells x 12 wells for 96 wells (96 holes). Taking Figures 1, 3, and 4 as examples, multiple sets are divided into 12 to 96 wells, with 2 wells in a set, 4 wells in a set, or 6 wells in a set.

[0114] The pharmacokinetic evaluation kit 1 may be covered with a lid 100 to prevent the intrusion of dust and germs.

[0115] This pharmacokinetic evaluation kit 1 may have one set of well plate 1a consisting of 2 to 6 wells and a passage as shown in FIGS. 1 to 4, or may have multiple sets of well plates 1a.

[0116] The insert containers 21-61, each having a cylindrical bottom covered with a porous plastic film 23-33, and the well plate 1a having wells 20-70, used to prepare the pharmacokinetic evaluation kit 1, are commercially available. The porous plastic films 23-33 seal the bottom of the insert containers 21-61 by heat fusion or adhesive bonding. Examples of the insert containers 21-61 include Transwell, Snapwell, and Falcon Culture Insert (all trade names manufactured by Corning Incorporated), and ad-MED Vitrigel (a trade name manufactured by Kanto Chemical Co., Inc.). The well plate 1a may be printed and molded using a 3D printer using a commercially available 3D printer ink composition containing acrylic resin, polystyrene resin, or acrylic resin, preferably acrylic resin, or may be molded from these resins, particularly acrylic resin. The upper rim of such insert containers has a flange that is wider than the upper rim of the wells. As a result, they are suspended so that the insert containers 21-61 are located in the center of the wells 20-60 when inserted into them. The cylindrical insert containers have a cylindrical lower half with the same diameter and an upper half that widens slightly toward the top and / or has ribs to prevent capillary action of the liquid culture medium between them and the side walls of the wells. Examples of cylindrical insert containers with such porous plastic films 23-33 attached, well plates 1a, and, if necessary, lids 100 that seal the wells to prevent the introduction of bacteria during culture include Transwell, Snapwell, Falcon Culture Insert (all trade names manufactured by Corning Incorporated), and ad-MED Vitrigel (trade name manufactured by Kanto Chemical Co., Inc.).

[0117] When preparing the hepatocyte culture membranes 23, 33, etc., hepatocytes are cultured in a conventional liquid or solid medium, preferably a commercially available or freshly prepared liquid medium, such as a liquid medium containing Dulbecco's modified Eagle's medium as a culture component and FBS (fetal bovine serum) as a conditioning component. An example of a liquid medium is commercially available DMEM (manufactured by Fujifilm Wako Pure Chemical Industries, Ltd.).

[0118] Such a liquid medium is more preferably supplemented with bile acids as an additive, since this further improves the orientation of the hepatocyte culture membrane. Bile acids are tetracyclic steroids widely found in the bile of mammals and have a basic structure in which one to three hydroxy groups are bound to a cholanic acid nucleus. Specific examples include primary bile acids such as cholic acid and chenodeoxycholic acid, secondary bile acids such as deoxycholic acid and lithocholic acid, which are converted by intestinal bacteria, and conjugated bile acids formed by amide bonds between these and glycine or taurine, such as taurocholic acid, taurochenodeoxycholic acid, glycodeoxycholic acid, tauroursodeoxycholic acid, and / or lithocholic acid. The bile acids may be of a single species or a plurality of species.

[0119] In the liquid medium, bile acids are contained at a concentration of, for example, preferably 1 μM to 300 μM, more preferably 100 μM.

[0120] To improve the orientation of hepatocyte culture membranes, the liquid medium may contain other additives in place of or in addition to bile acids, such as dimethyl sulfoxide (DMSO), differentiation promoters that induce the expression of enzymes or transporters, such as activated vitamin D, azacytidine, colchicine, rifampicin, and dexamethasone, and additives that induce orientation, such as vitamin A and cobalt chloride.

[0121] In the pharmacokinetic evaluation kit 1, it is difficult to store and maintain the membranous cell layers 23, 33, etc. for a long period of time. Therefore, it is preferable to prepare the cell culture membranes 23, 33, etc. and prepare the pharmacokinetic evaluation kit 1 immediately before measurement by the pharmacokinetic evaluation method.

[0122] The pharmacokinetic evaluation method using the pharmacokinetic evaluation kit 1 of FIGS. 1 to 4 is described below with reference to FIG. 3-1 as a representative example.

[0123] As shown in Figure 3-1, in the pharmacokinetic evaluation kit 1, the vascular test liquid 26 (see Figure 1) is poured into wells 20-40 and 70, communicating them via passages 10a-10c and 10e. Next, the gastrointestinal test liquid 25 is poured into the insert container 21 supporting the gastrointestinal cell culture membrane in well 20. The bile duct test liquid 35 is poured into the insert container 31 supporting the bile duct epithelial cell culture membrane in well 30. The renal tubule test liquid 45 is poured into the insert container 41 supporting the renal tubule cell culture membrane in well 40. Each insert container 21-41 is inserted into and suspended in each well 20-40, and the cell culture membrane is immersed in the vascular test liquid. The magnetic stirrer (not shown) is driven to rotate the circulator 78. The vascular test liquid then circulates through the wells 20-40 and becomes uniform and stable.

[0124] The basic composition of the vascular test solution 26, etc., and the gastrointestinal test solution 25, bile duct test solution 35, and renal tubule test solution 45 is a buffer solution. The buffer solution is not particularly limited as long as it does not damage the hepatocyte culture membrane, and examples thereof include phosphate buffer solution at pH 7.4, phosphate-buffered saline at pH 7.4, and physiological isotonic buffered salt solution (Hank's balanced salt solution: HBSS-HEPES (pH 7.4)).

[0125] When performing a pharmacokinetic evaluation method for a target drug administered to the gastrointestinal tract, bile duct, or renal tubules to determine whether the drug is easily metabolized and absorbed in the organs or easily distributed in the blood vessels, the target drug is added to the gastrointestinal cell culture membrane-supported insert container 21 in well 20 so that it dissolves in the gastrointestinal tract-side test fluid 25. After a short period of time, the concentrations of the gastrointestinal tract-side test fluid 25 in the gastrointestinal cell culture membrane-supported insert container 21 in well 20, the bile duct-side test fluid 35 in the bile duct epithelial cell culture membrane-supported insert container 31 in well 30, the renal tubule-side test fluid 45 in the renal tubule cell culture membrane-supported insert container 41 in well 40, and the blood vessel-side test fluid are measured. This makes it possible to measure the amount of the target drug moving through the cell culture membranes consisting of a membranous cell layer and a porous plastic film in each of the insert containers 21-41. The concentration can be measured and quantified by optical techniques, such as fluorescence intensity or transmittance or absorbance at a specific measurement wavelength such as ultraviolet light, or by high-performance liquid chromatography (HPLC) or liquid chromatography tandem mass spectrometry (LC-MS / MS). If necessary, quantification can be performed using a calibration curve of different standard concentrations and their transmittance or absorbance.

[0126] Regarding the pharmacokinetic evaluation method using this pharmacokinetic evaluation kit 1, an example of a series of steps including the steps of preparing insert containers 21 to 41 having cell culture membranes, the step of fabricating pharmacokinetic evaluation kit 1, and the subsequent pharmacokinetic evaluation step is described in more detail below.

[0127] The first step is to prepare insert containers 21 to 41 each having a cell culture membrane. Specifically, at the time of pharmacokinetic evaluation, a predetermined amount of hepatocytes is seeded on the bottom of well 30 of well plate 1a according to a standard method and cultured for several days to obtain hepatocytes in a cluster or layered form.

[0128] The next step is to prepare insert containers 21 to 41 having cell culture membranes. First, the preparation of insert container 31 will be described. Cells 32, which are hepatocytes such as hepatoma cells, e.g., HepG2 cells, are seeded on a porous plastic film 33 (see FIG. 1) provided at the bottom of an empty insert container tube 31 for containing bile duct-side test fluid 35, such as a Transwell (trade name manufactured by Corning Incorporated) insert container tube or an insert container using a collagen vitrigel membrane (Vitrigel is a registered trademark), and made of collagen-coated polytetrafluoroethylene with a pore size of 3.0 μm.

[0129] The plated cells 32 are cultured on the porous plastic film 33 in a conventional culture medium, such as DMEM, placed inside the tube of the insert container 31, while the insert container 31 is covered with a lid 100 (see FIG. 1) to prevent contamination by bacteria. One day later, the culture medium is replaced with a similar culture medium except that a bile acid, such as cholic acid, deoxycholic acid, taurocholic acid, chenodeoxycholic acid, taurochenodeoxycholic acid, glycodeoxycholic acid, tauroursodeoxycholic acid, or lithocholic acid, is added at a concentration of 100 μM. The cells 32 are cultured for 1 to 4 weeks, for example, up to the 7th, 14th, or 21st day. The culture medium is replaced with a similar bile acid-containing culture medium at an appropriate time, for example, the 4th, 7th, 10th, or 14th day, and the cells 32 are continued to be cultured. As the cells 32 grow, they grow into a cell culture membrane on the porous plastic film 33, forming a hepatocyte culture membrane 34.

[0130] The membranous cell layer in this hepatocyte culture membrane 34 is composed of cells 32, and is a monolayer membrane in which the cells 32 grow in a single layer with no gaps between them. This results in the formation of a hepatocyte culture membrane 34, consisting of a membranous cell layer of cells 32 and a porous plastic film 33. This membranous cell layer has an orientation such that the lower side of the membranous cell layer, i.e., the porous plastic film 33 side, faces the blood vessel, and the upper side of the membranous cell layer, i.e., the side opposite the porous plastic film 33, faces the organ. A test solution containing a drug to be measured, the drug's potential for biliary excretion from the liver or systemic uptake, is dissolved in the porous plastic film 33, and the drug freely permeates through both the front and back of the porous plastic film 33. However, due to the orientation of the membranous cell 32 layer, permeation is easier from the blood vessel side B to the organ side A than from the organ side A to the blood vessel side B. This results in the preparation of a hepatocyte culture membrane-supported insert container 31 having an oriented hepatocyte culture membrane.

[0131] The insert containers 21 and 41 are prepared in the same manner.

[0132] The other step is to prepare and prepare a well plate 1a. Specifically, an empty well plate 1a is prepared, which has wells 20-40 for inserting the cell culture membrane-supporting insert containers 21-41 and a well 70 for accommodating the circulatory system 78, and which is used to accommodate the blood vessel-side test solution into which the cell culture membranes of the cell culture membrane-supporting insert containers 21-41 are immersed during pharmacokinetic evaluation. Such a well plate 1a has multiple wells, for example, 6 wells, 12 wells, or 24 wells, connected by passages.

[0133] In this way, a pharmacokinetic evaluation kit 1 consisting of the cell culture membrane-supporting insert containers 21 to 41 and the well plate 1a is produced.

[0134] Subsequently, in order to evaluate pharmacokinetics, for example, drug transport capacity, a vascular transport amount measurement step and an organ transport amount measurement step are performed, followed by a drug transport capacity calculation step. Specifically, on the seventh day of culture, which is the time to use the drug transport capacity evaluation method, the culture medium accumulated in the hollow spaces of the cell culture membrane-supporting insert containers 21-41 and the culture medium accumulated in the wells 20-40 / 70 are removed.

[0135] The vascular transport measurement step is carried out as follows: A vascular test liquid 26 consisting of only a buffer solution containing no drug to be measured is prepared. The vascular test liquid 26 is poured into well 20, passing through passages 10a to 10c and 10e until it reaches wells 30, 40, and 70. Next, a drug to be measured for pharmacokinetics is added to a buffer solution to prepare a gastrointestinal side test fluid 25. The gastrointestinal side test fluid 25 containing the drug to be measured for pharmacokinetics is slowly poured into and placed in a gastrointestinal cell culture membrane-supported insert container 21, taking care not to detach the cells 22 in the membranous cell layer. A bile duct side test fluid 35 and a renal tubule side test fluid 45 consisting only of a buffer solution without the drug to be measured are prepared, and these are slowly poured into a bile duct epithelial cell culture membrane-supported insert container 31 and a renal tubule cell culture membrane-supported insert container 41, respectively, taking care not to detach the cells 32 and 42 in the membranous cell layer. These insert containers 21-41 are inserted into and suspended in wells 20-40. The circulator 78 is gently rotated to circulate the vascular side test fluid 26.

[0136] Thereafter, the amount of the target drug transported between the vascular side test fluid and the organ side test fluid in each well is measured. The concentration of the target drug in the vascular side test fluid and the organ side test fluid in the insert containers 21 to 41 are measured to quantify the amount transported to the vascular side. For example, if the target drug is Rho123, a fluorescent substance that is a standard substrate for the transporter P-gp, the concentration can be measured from the amount of fluorescence.

[0137] Next, if necessary, the organ-side transport amount measurement step is performed as follows. Another cell culture membrane-supporting insert container 21-41 and well plate 1a are prepared. Another gastrointestinal tract-side test liquid 25, bile duct-side test liquid 35, and renal tubule-side test liquid 45 are prepared, each consisting of a buffer solution containing no target drug. These are slowly poured into the cell culture membrane-supporting insert containers 21-41, respectively, taking care not to detach the cells 22-42 from the membranous cell layer. Meanwhile, another vascular-side test liquid 25 is prepared by adding a target drug for pharmacokinetic measurement to a buffer solution. This vascular-side test liquid 25 is poured into the well 20 of the well plate 1a and placed therein, and the cell culture membranes of the cell culture membrane-supporting insert containers 21-41 are immersed in the vascular-side test liquid 25. The transport amount of the target drug from the vascular side B to the organ side A is then measured in the same manner. The transport amount can be measured by high performance liquid chromatography using a concentration-area calibration curve, by LC-MS / MS, or by colorimetry, such as colorimetry based on fluorescence absorption. Radioisotope measurements are also possible. The measurement sensitivity depends on the measurement method.

[0138] Finally, the drug transport capacity calculation step is carried out as follows: For each of wells 20 to 40, the ratio of the amount of the target drug transported from organ side A to blood vessel side B to the amount of the target drug transported from blood vessel side B to organ side A is calculated. Specifically, for multiple cases (e.g., n=3), the amount of the target drug transported from organ side A to blood vessel side B and the amount of the target drug transported from blood vessel side B to organ side A are measured at predetermined intervals, e.g., every 15 minutes, and a linear approximation equation with the horizontal axis representing time and the vertical axis representing the amount of transported from organ side A to blood vessel side B is calculated using a slope AtoB The transport speed per unit area of ​​the cell (cm / sec) was calculated by calculating the slope of the linear approximation equation, where the horizontal axis is time and the vertical axis is the transport amount from the blood vessel side B to the organ side A. BtoA The transport velocity per unit area of ​​the cell (cm / sec) is calculated by calculating the ratio of the slopes of the two. BtoA ) / (slope AtoB ) is calculated to determine the drug transport capacity of the drug being measured as pharmacokinetics. [Example]

[0139] Examples of the present invention will be described in detail below, but the scope of the present invention is not limited to these examples.

[0140] Example 1 Commercially available Caco-2 cells derived from ATCC were cultured on a vitrigel membrane in an insert container 21 manufactured by Kanto Chemical Co., Inc., using ad-MED Vitrigel 2 (trade name), and were cultured for 21 or 22 days, using a commercially available culture medium and changing the medium every 2 to 3 days, as an example of a known culture method. Bile duct organoids, which were established from primary human hepatocytes (PHH) capable of long-term proliferation and available, were also cultured on a vitrigel membrane in an insert container 31, using 1.0 × 10 cells in 40 μL / well, as an example of a known culture method. 4 Cells were seeded at a density of 1000 cells / well and incubated in a known medium at 37°C, 5% CO2, and 95% air for 7 to 10 days. Established organoids were collected with a pipette tip, and only bile duct organoids were selected based on their morphological differences. Single cell dissociation and passage were performed using TrypLE Express Enzyme. Bile duct organoids (50 cells / µL to 500 cells / µL) were suspended in 10% Matrigel medium and cultured for 12 or 13 days.

[0141] Next, a commercially available 24-well well plate (approximately 15 mmφ and 1.8 mm deep per well) was used as a mold, and a commercially available acrylic resin-containing ink composition was used to print and mold four of the 24 wells into a set as shown in Figure 3-1 using a 3D printer to create well plate 1a. In this well plate 1a, an insert container 21 containing Caco-2 cells was suspended in well 20, an insert container 31 containing bile duct organoid cells was suspended in well 30, well 40 was an empty well without suspending insert container 41, and well 70 was a circulation well containing a magnetic stirrer bar, which is a circulator 78, without suspending an insert container. Well plate 1a was placed on a magnetic stirrer (not shown), and circulator 78 was rotated. Note that in Example 1, cells 37 in Figure 3-1 were not present.

[0142] A medium containing 10 μM rhodamine 123 (Rho123) (1% or 0.1% DMSO) as the target drug for pharmacokinetic evaluation was added to the insert container 21, which is the donor side (organ side A) of the well 20, at 37°C. In addition, the receiver side (blood vessel side B) of wells 20 to 40 and well 70 was incubated with a medium containing an equal concentration of DMSO.

[0143] For transport studies in the presence of inhibitors, medium containing 30 μM verapamil was added to both the organ and vascular sides. Up to 120 minutes or 24 hours after the start of the study, samples were collected from the vascular side B outside the insert container 21 of well 20 and the organ side A inside the insert container 31 of well 30, and replaced with an equal volume of medium. After sample collection, the medium was stirred in a 37°C, 5% CO2 incubator until the next sample collection time. The Rho123 concentration of the samples was measured using a plate reader. Based on the concentration results, orientation was analyzed using the above equations (1) and (2).

[0144] A schematic overview of the pharmacokinetics of well 20 and well 30 in this case is shown in Figure 5. As shown in Figure 5, the target drug moves from the luminal side (upper side, see well 20 in Figure 3-1), which is the organ side, of the gastrointestinal cells on the insert container 21 to the vascular side B, then passes through the circulating vascular side A, and moves to the luminal side Ai (upper side, see well 30 in Figure 3-1) of the bile duct epithelial cells in the insert container 31. However, when an inhibitor is added, more of the target drug moves from the luminal side (upper side, see well 20 in Figure 3-1) of the gastrointestinal cells on the insert container 21 to the vascular side B, passes through the circulating vascular side A, and reaches the well 30 in which the insert container 31 with the bile duct epithelial cells attached is suspended, but moves less to the luminal side Ai (upper side, see well 30 in Figure 3-1) of the bile duct epithelial cells in the insert container 31.

[0145] The results are shown in Table 1 and Figure 6. As is clear from Table 1 and Figure 6, according to the drug transport evaluation kit of Example 1 and the drug transport evaluation method using it, the inhibitor inhibits the release protein in well 20, causing side B to increase, and in well 30, the inhibitor causes a large amount of the target drug to remain on side B, indicating whether or not the target drug is excreted in bile after being absorbed in the digestive tract and entering the blood circulation. (In Table 1, Papp A to B is higher in the presence of verapamil than in the control, and the results of Figure 1 and Table 1 indicate that rhodamine 123, the target drug whose pharmacokinetics should be evaluated, is excreted in bile.)

[0146] [Table 1]

[0147] [Table 2]

[0148] Furthermore, as shown in the results in Table 2, the concentration of rhodamine 123, the target drug for pharmacokinetic evaluation, varied only to a degree of error in each well, indicating that it circulated evenly.

[0149] Thus, the drug transport evaluation kit and the drug transport evaluation method using it have been demonstrated to be useful as an ex vivo test model, despite being an in vitro experimental system. In particular, it has been confirmed that it can be used as a test model that corresponds to in vivo kinetics, especially in humans, and allows for appropriate, simple, and rapid investigation. [Industrial Applicability]

[0150] The drug transport evaluation kit of the present invention and the drug transport evaluation method using the same can be used to easily examine and accurately predict the drug transport ability in various organs and the pharmacokinetics such as the pharmacokinetics and toxicity of a drug when an administered drug circulates throughout the body while moving from the organ side to the blood vessel side or from the blood vessel side to the organ side. [Explanation of symbols]

[0151] 1 is a pharmacokinetic evaluation kit, 1a is a well plate, 10a, 10b, 10c, 10d, 10e, 10f, and 10g are passages, 20 is a well, 21 is an insert container, 21a is an upper flange of the insert container, 22 is organ-derived cells, 23 is a porous plastic film, 24 is a membranous cell culture membrane, 25 is an organ-side test fluid, 26 is a blood vessel-side test fluid, 27 is a mass or layer of cells, 30 is a well, 31 is an insert container, 31a is an upper flange of the insert container, 32 is an organ-derived cell, and 33 is a porous plastic film, 34 membranous cell culture membrane, 35 organ-side test fluid, 36 blood vessel-side test fluid, 37 clumped or layered cells, 40 well, 41 insert container, 42 organ-derived cells, 45 organ-side test fluid, 50 well, 52 organ-derived cells, 56 blood vessel-side test fluid, 60 well, 61 insert container, 62 organ-derived cells, 65 organ-side test fluid, 70 well, 75 blood vessel-side test fluid, 78 circulatory system, 100 lid, 110 water bath, 111 warm water, 112 spring net A 20 A 30 is the organ side, B 20 ·B 30 is the vascular side.

Claims

1. A pharmacokinetic evaluation kit characterized by having a well plate with multiple recessed wells connected by a passageway midway in the depth direction for holding a test liquid containing a target drug whose pharmacokinetics is to be evaluated using organ-derived cells.

2. The pharmacokinetic evaluation kit according to claim 1, characterized in that it comprises a well plate in which the plurality of wells are connected by the passages to form a flow path for diffusing, circulating, and / or circulating the test liquid.

3. 2. The pharmacokinetic evaluation kit according to claim 1, further comprising: a well plate; and a membranous cell culture membrane at the bottom, in which the organ-derived cells are arranged closely together on a porous plastic film in the form of a single layer or in the form of a mass or laminate in which the organ-derived cells are closely overlapped; an insert container for holding an organ-side test liquid and being inserted into and suspended in at least one of the plurality of wells to immerse the membranous cell culture membrane in the blood vessel-side test liquid; wherein the organ-derived cells have a drug transport orientation such that the organ-derived cells are on the blood vessel side below the membranous cell culture membrane and on the organ side above the membranous cell culture membrane.

4. The pharmacokinetic evaluation kit according to claim 3, characterized in that the insert containers containing different organ-derived cells in the order of blood circulating organs and / or organs in the order of in vivo kinetics of the drug to be measured are inserted and suspended in the multiple wells in sequence from upstream to downstream via the passage, and the wells are connected linearly, radially, or circularly via the passage.

5. 5. The pharmacokinetic evaluation kit according to claim 4, wherein cultured cells in a monolayer or non-monolayer membrane form are present on the bottom of at least one of the wells on the upstream side.

6. The pharmacokinetic evaluation kit according to claim 1, characterized in that the flow path connecting the plurality of wells is parallel to the shortest distance line between the wells in the horizontal direction, or is inclined at a maximum of 45° in the horizontal direction from the shortest distance line.

7. 2. The pharmacokinetic evaluation kit according to claim 1, wherein the well plate has a water bath for heating at a constant temperature or is placed in a thermostatic bath.

8. The pharmacokinetic evaluation kit according to claim 1, characterized in that the plurality of wells have one or more depressions at the bottom of each well, each depression having a diameter of 100 μm to 1 mm and a depth of 20 μm to 40 μm, or the plurality of wells themselves serve as the depressions.

9. 5. The pharmacokinetic evaluation kit according to claim 4, wherein the plurality of wells are connected in a circular fashion, and at least one of the wells has a circulator that circulates the blood vessel-side test liquid through the flow path.

10. The pharmacokinetic evaluation kit according to claim 9, wherein the circulatory organ is a stirrer bar.

11. 11. The pharmacokinetic evaluation kit according to claim 10, wherein the stir bar has a neodymium magnet.

12. 2. The pharmacokinetic evaluation kit according to claim 1, wherein the organ-derived cells are hepatocytes, gastrointestinal cells, bile duct epithelial cells or bile duct cells, renal tubular cells, cerebrovascular endothelial cells, vascular endothelial cells, or placental cells, which can be seeded in the form of a monolayer membrane, or hepatocytes, cardiac muscle cells, lung-derived cells, smooth muscle cells, or bile duct epithelial cells or bile duct cells, which can be seeded in the form of a mass or layer.

13. As the organ-derived cells, the hepatocytes are at least any of hepatoma cells, free hepatocytes, fresh hepatocytes derived from animals or humans, iPS cells and / or ES cells induced to hepatocytes, spheroids and / or organoids of hepatocytes, and immortalized hepatocytes; the gastrointestinal cells are at least any one of gastrointestinal cancer cells, free gastrointestinal cells, fresh gastrointestinal cells derived from an animal or human, iPS cells and / or ES cells induced to become gastrointestinal cells, spheroids and / or organoids of gastrointestinal cells, and immortalized gastrointestinal cells; the bile duct epithelial cells or bile duct cells are at least any of cholangiocarcinoma cells, free bile duct epithelial cells, fresh bile duct epithelial cells derived from an animal or a human, iPS cells and / or ES cells induced to become bile duct epithelial cells or bile duct cells, spheroids and / or organoids of bile duct epithelial cells or bile duct cells, and immortalized bile duct epithelial cells or bile duct cells, the renal tubular cells are at least any of renal tubular cancer cells, free renal tubular cells, fresh renal tubular cells derived from an animal or human, iPS cells and / or ES cells induced to renal tubular cells, spheroids and / or organoids of renal tubular cells, and immortalized renal tubular cells; the cerebrovascular endothelial cells are at least any one of cerebrovascular endothelial cancer cells, free cerebrovascular endothelial cells, fresh cerebrovascular endothelial cells derived from animals or humans, iPS cells and / or ES cells induced to cerebrovascular endothelial cells, spheroids and / or organoids of cerebrovascular endothelial cells, and immortalized cerebrovascular endothelial cells; the vascular endothelial cells are at least any one of vascular endothelial cancer cells, free vascular endothelial cells, fresh vascular endothelial cells derived from animals or humans, iPS cells and / or ES cells induced to become vascular endothelial cells, spheroids and / or organoids of vascular endothelial cells, and immortalized vascular endothelial cells; the placental cells are at least any of free placental cells, animal-derived humanized fresh placental cells, iPS cells and / or ES cells induced from placental cells, spheroids and / or organoids of placental cells, and immortalized placental cells; the cardiomyocytes are at least any of myocardial cancer cells, free cardiomyocytes, fresh cardiomyocytes derived from animals or humans, iPS cells and / or ES cells induced into cardiomyocytes, spheroids and / or organoids of cardiomyocytes, and immortalized cardiomyocytes; the lung-derived cells are at least any of lung-derived cancer cells, free lung-derived cells, fresh lung-derived cells derived from an animal or human, iPS cells and / or ES cells induced from lung-derived cells, spheroids and / or organoids of lung-derived cells, and immortalized lung-derived cells; The pharmacokinetic evaluation kit according to claim 7, characterized in that the smooth muscle cells are at least any of smooth muscle-derived cancer cells, free smooth muscle-derived cells, fresh smooth muscle-derived cells derived from animals or humans, iPS cells and / or ES cells induced into smooth muscle-derived cells, spheroids and / or organoids of smooth muscle-derived cells, and immortalized smooth muscle-derived cells.

14. The pharmacokinetic evaluation kit described in claim 3, characterized in that the insert container, which has a monolayered membranous cell culture membrane made of the organ-derived cells on the porous plastic film at its bottom, and another insert container, which has a clumped or layered membranous cell culture membrane made of another organ-derived cell on another porous plastic film at its bottom, are inserted into and suspended in at least one of the multiple wells.

15. 6. The pharmacokinetic evaluation kit according to claim 5, wherein the cultured cells are hepatocytes, cardiac muscle cells, lung-derived cells, or smooth muscle cells that can be seeded in the non-monolayer form of a clump or laminated membrane.

16. The cultured cells include: the hepatocytes are at least any of hepatoma cells, free hepatocytes, fresh hepatocytes derived from animals or humans, iPS cells and / or ES cells induced to hepatocytes, spheroids and / or organoids of hepatocytes, and immortalized hepatocytes; the cardiomyocytes are at least any of myocardial cancer cells, free cardiomyocytes, animal-derived humanized fresh cardiomyocytes, iPS cells and / or ES cells induced into cardiomyocytes, cardiomyocyte spheroids and / or organoids, and immortalized cardiomyocytes; The pharmacokinetic evaluation kit of claim 15, characterized in that the lung-derived cells are at least any of lung-derived cancer cells, free lung-derived cells, fresh lung-derived cells derived from animals or humans, iPS cells and / or ES cells induced from lung-derived cells, spheroids and / or organoids of lung-derived cells, and immortalized lung-derived cells.

17. A pharmacokinetic evaluation method using the pharmacokinetic evaluation kit according to claim 1, characterized in that pharmacokinetics is evaluated by culturing cells derived from different organs in at least two or more of the plurality of wells, adding a target drug to be measured, and measuring the concentration of the target drug in the test solution in the plurality of wells via the passage.

18. 18. The pharmacokinetic evaluation method according to claim 17, wherein the test liquid is diffused, circulated, and / or circulated within a flow path in which the plurality of wells are connected by the passage.

19. 18. The method for pharmacokinetic evaluation according to claim 17, wherein the organ-derived cells are arranged closely on a porous plastic film to form a single layer or a clump or stack of cells closely overlapping each other to form a membranous cell culture membrane at the bottom of an insert container, and then the insert containers are inserted into and suspended in the multiple wells with an organ-side test liquid placed therein, and the membranous cell culture membrane is immersed in the blood vessel-side test liquid, which is the test liquid, so that the organ-derived cells have a drug transport orientation such that the organ-derived cells are on the blood vessel side below the membranous cell culture membrane and on the organ side above the membranous cell culture membrane, and then the drug to be measured is added to the upstream well or the insert container, and the concentrations of the drug to be measured in the blood vessel-side test liquid in the well and the organ-side test liquid in the insert container are measured, the transport amount of the drug to be measured is calculated over time, and the transport rate is analyzed to evaluate the pharmacokinetics.

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