Drug evaluation method, reagent, and kit

Abstract

The purpose of the present invention is to provide a method for evaluating a drug using myocardial cells and with which it is possible to stably evaluate the effect of a drug such as a muscarinic receptor agonist, an adrenergic receptor inhibitor, and a muscarinic receptor inhibitor on the heart rate. And a reagent and a kit for performing the method for evaluating the drug. According to the present invention, provided is a method for evaluating a drug, comprising: a step for treating myocardial cells with a culture medium containing a physiologically active substance of the sympathetic nervous system and / or a physiologically active substance of the parasympathetic nervous system; a step in which a drug is brought into contact with the treated myocardial cells; and evaluating the influence of the drug on the heart rate of the myocardial cells.

Description

Technical Field

[0001] The present invention relates to a method for evaluating a drug, comprising the following steps:

[0002] The effects of drugs on the heart rate of cardiomyocytes are evaluated using cardiomyocytes. The present invention also relates to a reagent and a kit for performing the above-mentioned drug evaluation method. Background Art

[0003] One method for diagnosing heart disease is to measure the beating frequency of cardiomyocytes. Furthermore, for the purpose of drug screening, drug efficacy or toxicity evaluation is performed using cardiomyocytes differentiated from pluripotent stem cells or primary cardiomyocytes established from living organisms.

[0004] For example, Patent Document 1 describes the ability to evaluate increases and decreases in heart rate caused by adrenergic receptor agonists or muscarinic receptor agonists using cardiomyocytes derived from primate pluripotent stem cells. Non-Patent Document 1 describes the use of primary cardiomyocytes derived from mice to demonstrate a decrease in heart rate caused by a muscarinic receptor agonist and an increase in heart rate caused by a muscarinic receptor inhibitor. Furthermore, Non-Patent Document 2 describes the use of cardiomyocytes derived from iPS cells to evaluate drugs and describes a decrease in heart rate caused by a muscarinic receptor agonist.

[0005] Patent Document 2 describes the differentiation of human embryonic stem (hES) cells into cardiomyocytes by co-culturing them with cells that secrete cardiomyocyte differentiation-deriving factors or an extracellular medium obtained therefrom under differentiation-deriving conditions, and also describes testing the effects of phenylephrine, isoproterenol, and carbachol on cultured isolated hES cardiomyocytes.

[0006] Patent Document 3 describes a method for producing a contractile cell construct, comprising the steps of: seeding preselected cells containing signal-releasing particles onto a silicone mold; and culturing the preselected cells to produce the contractile cell construct.

[0007] Prior art literature

[0008] Patent Literature

[0009] Patent Document 1: Japanese Patent No. 5917429

[0010] Patent Document 2: Japanese Patent Application No. 2006-523091

[0011] Patent Document 3: U.S. Patent Application Publication No. 2018 / 0044640

[0012] Non-patent literature

[0013] Non-patent document 1: Kohashi et al., Biological Rhythm Research, 34(4), 367-381, 2004

[0014] Non-patent literature 2: Lu et al., Frontiers in Physiology, 13, Article 838435, 2022 Summary of the Invention

[0015] Technical issues to be solved by the invention

[0016] The present inventors' research has clearly shown that conventional evaluation methods using cardiomyocytes can make it difficult to evaluate muscarinic receptor agonists. Furthermore, conventional evaluation methods using cardiomyocytes have clearly shown that inhibitors of adrenergic and muscarinic receptors cannot be evaluated.

[0017] The present invention aims to provide a method for evaluating drugs using cardiomyocytes, capable of high-throughput evaluation of the effects of drugs such as muscarinic receptor agonists, adrenergic receptor inhibitors, and muscarinic receptor inhibitors on heart rate. Another object of the present invention is to provide a reagent and kit for use in the aforementioned drug evaluation method.

[0018] Means for solving technical problems

[0019] The present inventors conducted intensive research to address the above-mentioned issues and have discovered that, in a method for contacting cardiomyocytes with a drug and evaluating the drug's effect on the cardiomyocyte's heart rate, the cardiomyocytes are treated with a culture medium containing a physiologically active substance of the sympathetic nervous system and / or a physiologically active substance of the parasympathetic nervous system prior to contact with the drug, thereby enabling high-throughput drug evaluation. The present invention has been completed based on this finding.

[0020] That is, according to the present invention, the following inventions are provided.

[0021] <1> A method for evaluating a drug, comprising the following steps:

[0022] a step of treating cardiomyocytes with a culture medium containing a physiologically active substance of the sympathetic nervous system and / or a physiologically active substance of the parasympathetic nervous system;

[0023] a step of contacting a drug with the cardiomyocytes treated above; and

[0024] Steps for evaluating the effects of drugs on the heart rate of cardiomyocytes.

[0025] <2> The method according to <1>, wherein

[0026] The above culture medium is serum-free.

[0027] <3> The method according to <1> or <2>, wherein

[0028] The physiologically active substances of the sympathetic nervous system include adrenaline receptor agonists.

[0029] <4> The method according to <1> or <2>, wherein

[0030] The physiologically active substances of the parasympathetic nervous system include agonists of muscarinic receptors.

[0031] <5> The method according to <1> or <2>, comprising the following steps:

[0032] The inhibitory effects of the above drugs on the above adrenaline receptors were evaluated.

[0033] <6> The method according to <1> or <2>, comprising the following steps:

[0034] The inhibitory effects of the above drugs on the above muscarinic receptors were evaluated.

[0035] <7> The method according to <1> or <2>, wherein

[0036] The aforementioned cardiomyocytes are cardiomyocytes derived from human iPS cells.

[0037] <8> The method according to <1> or <2>, wherein

[0038] The physiologically active substance of the parasympathetic nervous system is carbachol, and the physiologically active substance of the sympathetic nervous system is isoproterenol.

[0039] <9> A reagent for performing the method described in <1> or <2>, the reagent comprising an adrenaline receptor agonist and / or a muscarinic receptor agonist.

[0040] <10> A kit comprising the reagent according to <9> and a culture medium.

[0041] <11> The kit according to <10>, further comprising cardiomyocytes.

[0042] Effects of the Invention

[0043] According to the present invention, the effects of adrenaline and muscarinic receptor agonists and adrenaline and muscarinic receptor inhibitors on heart rate can be evaluated at a high throughput using cardiomyocytes. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Indicates the measurement result of the beat time (seconds).

[0045] Figure 2 The results show the evaluation of the effect of adding a muscarinic receptor agonist (carbachol) to a serum-free medium on heart rate.

[0046] Figure 3 The results are shown, which show the results of evaluating the decrease in heart rate caused by a muscarinic receptor agonist (carbachol) using a serum-free medium supplemented with an adrenergic receptor agonist (isoproterenol).

[0047] Figure 4 The results are shown, which show the results of evaluating the heart rate reduction caused by a muscarinic receptor agonist (cevimeline) using a serum-free medium supplemented with an adrenergic receptor agonist (isoproterenol).

[0048] Figure 5 The results are shown, which show the results of evaluating the decrease in heart rate caused by a muscarinic receptor agonist (pilocarpine) using a serum-free medium supplemented with an adrenergic receptor agonist (isoproterenol).

[0049] Figure 6 The results of evaluating the reduction in heart rate caused by carbachol using a maintenance medium are shown.

[0050] Figure 7 The results are shown, which show the results of evaluating the heart rate increase caused by a muscarinic receptor inhibitor (atropine) using a serum-free culture medium supplemented with an adrenergic receptor agonist (isoproterenol) and a muscarinic receptor agonist (carbachol).

[0051] Figure 8 The results show the results of evaluating the heart rate increase caused by a muscarinic receptor inhibitor (tiotropium bromide) using a serum-free culture medium supplemented with an adrenergic receptor agonist (isoproterenol) and a muscarinic receptor agonist (carbachol).

[0052] Figure 9 The results are shown, which show the results of evaluating the heart rate increase caused by a muscarinic receptor inhibitor (glycopyrrolate) using a serum-free culture medium supplemented with an adrenergic receptor agonist (isoproterenol) and a muscarinic receptor agonist (carbachol).

[0053] Figure 10 The results show the results of evaluating the heart rate reduction caused by an adrenergic receptor inhibitor (propranolol) using a serum-free culture medium supplemented with an adrenergic receptor agonist (isoproterenol) and a muscarinic receptor agonist (carbachol).

[0054] Figure 11 The results show the evaluation of the heart rate reduction caused by an adrenergic receptor inhibitor (atenolol) using a serum-free culture medium supplemented with an adrenergic receptor agonist (isoproterenol) and a muscarinic receptor agonist (carbachol).

[0055] Figure 12 The results show the results of evaluating the effect of adding a drug (aspirin) that has no effect on heart rate to a serum-free culture medium to which an adrenergic receptor agonist (isoproterenol) and a muscarinic receptor agonist (carbachol) were added.

[0056] Figure 13 The present invention shows the method and results of evaluating the heart rate increase caused by a muscarinic receptor inhibitor (atropine) using a maintenance medium.

[0057] Figure 14 This paper shows the method and results of evaluating the heart rate increase caused by a muscarinic receptor inhibitor (atropine) using a serum-free medium. DETAILED DESCRIPTION

[0058] The present invention is described in detail below. In this specification, the numerical range represented by "to" means that the numerical values ​​before and after "to" are included in the range as the minimum and maximum values, respectively. The meaning of μM is the same as the meaning of μmol / L.

[0059] The method for evaluating the drug of the present invention includes the following steps: a step of treating myocardial cells with a culture medium containing physiologically active substances of the sympathetic nervous system and / or physiologically active substances of the parasympathetic nervous system; a step of bringing the drug into contact with the treated myocardial cells; and a step of evaluating the effect of the drug on the heart rate of the myocardial cells.

[0060] The cardiomyocytes in the present invention may be either cardiomyocytes derived from pluripotent stem cells or cardiomyocytes established from tissues (preferably primary cardiomyocytes), but are preferably cardiomyocytes derived from pluripotent stem cells.

[0061] Pluripotent stem cells refer to cells with the pluripotency (pluripotency) of all cells constituting a living body and the self-replication ability of the cells that can maintain their pluripotency even after cell division. As pluripotent stem cells, induced pluripotent stem cells (iPS cells), embryonic stem cells (ES cells) or embryonic germ cells (EG cells) can be enumerated. Preferably, pluripotent stem cells are induced pluripotent stem cells (iPS cells) or embryonic stem cells (ES cells). That is, as the cardiomyocyte in the present invention, preferably the cardiomyocyte derived from induced pluripotent stem cells (iPS cells) or the cardiomyocyte derived from embryonic stem cells (ES cells), more preferably the cardiomyocyte derived from iPS cells.

[0062] The biological species of the cardiomyocytes is not particularly limited, but mammals are preferred, rodents (mice, rats, hamsters, etc.) or primates (monkeys, humans, etc.) are more preferred, primates are further preferred, and humans are particularly preferred.

[0063] The cardiomyocytes are particularly preferably those derived from human iPS cells.

[0064] Methods for deriving cardiomyocytes from pluripotent stem cells are known, and examples thereof include the methods described in International Application Publication No. 2013 / 111875, International Application Publication No. 2015 / 182765, International Application Publication No. 2020 / 027278, and Minami I et al., Cell Rep. 2012 Nov 29; 2(5): 1448-60. Furthermore, cardiomyocytes derived from pluripotent stem cells may be commercially available cardiomyocytes derived from ES cells or iPS cells (e.g., iCell (registered trademark) Cardiomyocytes (manufactured by FUJIFILM Cellular Dynamics, Inc.) and iCell (registered trademark) Cardiomyocytes 2.0 (manufactured by FUJIFILM Cellular Dynamics, Inc.)).

[0065] The cardiomyocytes used in the present invention are cultured cells, preferably cultured in a two-dimensional manner, preferably in a two-dimensional sheet. That is, the cardiomyocytes used in the present invention are preferably not three-dimensional organoids. Using cultured cells in a two-dimensional manner rather than organoids allows for simpler drug evaluation.

[0066] The cardiomyocytes used in the present invention preferably include ventricular cells, atrial cells, and nodal cells. Furthermore, as a type of cardiomyocyte, cells called pacemaker cells are known, but pacemaker cells are mostly (90-100%) composed of nodal cells. The cardiomyocytes used in the present invention are preferably cells composed mainly of ventricular cardiomyocytes, and are different from pacemaker cells.

[0067] The proportion of nodal cells in the cardiomyocytes used in the present invention is preferably 80% or less, more preferably 50% or less, further preferably 30% or less, and may be 20% or less.

[0068] The proportion of cells expressing ventricular markers (ie, ventricular cardiomyocytes) in the cardiomyocytes used in the present invention is preferably 30% or more, more preferably 40% or more, and even more preferably 50% or more.

[0069] For example, ventricular cardiomyocytes, cardiac cardiomyocytes, and nodal cardiomyocytes can be distinguished based on the shape of their action potentials. An example of this is the method described in Ma J. et al. Am J Physiol Heart Circ Physiol 301: H2006-H2017 (2011). Furthermore, differentiation can be achieved by immunostaining using the expression of specific proteins as markers. In this case, MLC-2V can be used as a marker for ventricular cardiomyocytes, MLC-2a can be used as a marker for atrial cells, and HCN can be used as a marker for nodal cells.

[0070] In the present invention, the cardiomyocytes can be pre-cultured before being treated with a culture medium containing physiologically active substances of the sympathetic nervous system and / or physiologically active substances of the parasympathetic nervous system. For example, when using frozen cardiomyocytes, the frozen cardiomyocytes can be thawed and suspended in a thawing medium (for example, when using iCell Cardiomyocytes 2.0 manufactured by FUJIFILM Cellular Dynamics, Inc. as cardiomyocytes, they can be suspended in iCell Cardiomyocyte Thawing Medium manufactured by FUJIFILM Cellular Dynamics, Inc.), and then seeded on a plate at 10,000 to 100,000 cells per well. After the cardiomyocytes are allowed to stand in a CO2 incubator, a maintenance medium is added (for example, when iCell cardiomyocytes 2.0 manufactured by FUJIFILM Cellular Dynamics, Inc. are used as cardiomyocytes, iCell cardiomyocyte maintenance medium manufactured by FUJIFILM Cellular Dynamics, Inc. is added). The cells can be cultured in a CO2 incubator for 1 day to 4 weeks, preferably 2 days to 2 weeks. In addition, the culture medium can be replaced with a serum-free medium the day before the cardiomyocytes are treated with a culture medium containing physiologically active substances of the sympathetic nervous system and / or physiologically active substances of the parasympathetic nervous system.

[0071] In the present invention, the cardiomyocytes are treated with a culture medium containing a physiologically active substance of the sympathetic nervous system and / or a physiologically active substance of the parasympathetic nervous system before the drug is brought into contact with the cardiomyocytes. The culture medium preferably contains both physiologically active substances of the sympathetic nervous system and physiologically active substances of the parasympathetic nervous system.

[0072] As physiologically active substances of the sympathetic nervous system, there are adrenaline receptor agonists, and examples thereof include epinephrine, norepinephrine, isoproterenol, and terbutaline.

[0073] As physiologically active substances of the parasympathetic nervous system, there are agonists of muscarinic receptors, and examples thereof include acetylcholine, muscarine, carbachol, bethanechol, cevimeline, and pilocarpine.

[0074] When an adrenaline receptor agonist is used, the concentration of the adrenaline receptor agonist in the culture medium is preferably 0.05 μmol / L to 50 μmol / L, more preferably 0.1 μmol / L to 20 μmol / L, further preferably 0.5 μmol / L to 10 μmol / L, particularly preferably 0.5 μmol / L to 5 μmol / L, and most preferably 0.5 μmol / L to 2 μmol / L.

[0075] When a muscarinic receptor agonist is used, the concentration of the muscarinic receptor agonist in the culture medium is preferably 0.1 μmol / L to 100 μmol / L, more preferably 0.5 μmol / L to 50 μmol / L, further preferably 1 μmol / L to 20 μmol / L, particularly preferably 1 μmol / L to 10 μmol / L, and most preferably 1 μmol / L to 5 μmol / L.

[0076] When both physiologically active substances of the sympathetic nervous system and physiologically active substances of the parasympathetic nervous system are used simultaneously, the ratio of the concentrations of physiologically active substances of the sympathetic nervous system and physiologically active substances of the parasympathetic nervous system in the culture medium is preferably 0.1:1 to 1:20, more preferably 0.5:1 to 1:10, further preferably 1:1 to 1:5, and as an example, 1:3.

[0077] The culture time for treating cardiomyocytes with a culture medium containing physiologically active substances of the sympathetic nervous system and / or parasympathetic nervous system is generally 1 minute to 4 hours, preferably 5 minutes to 2 hours, more preferably 10 minutes to 1 hour, further preferably 10 minutes to 30 minutes.

[0078] As a culture medium to which physiologically active substances of the sympathetic nervous system and / or the parasympathetic nervous system are added, it is not particularly limited as long as it is a culture medium commonly used for the cultivation of cardiomyocytes. However, in the case of serum-free or serum-containing, the content of serum is preferably 3% by volume or less, more preferably 1% by volume or less. As a culture medium, a serum-free culture medium is particularly preferred, and a culture medium that does not contain albumin can also be used. As a culture medium to which physiologically active substances of the sympathetic nervous system and / or the parasympathetic nervous system are added, for example, a serum-free culture medium for detecting cardiomyocytes manufactured by FUJIFILM Cellular Dynamics, Inc. can be cited, but it is not particularly limited.

[0079] The container for culturing cardiomyocytes is not particularly limited as long as it is a container commonly used for cell culture. The shape of the container can include a culture dish, a flat plate, a bottle, a chamber, a multi-well plate (for example, a 6-well plate, a 12-well plate, a 24-well plate, a 48-well plate, a 96-well plate, or a 384-well plate). Among the above, a multi-well plate is preferred, and from the viewpoint of high throughput, a 48-well plate or a 96-well plate is particularly preferred.

[0080] The cardiomyocyte culture format is preferably planar culture.

[0081] Regarding the number of cardiomyocytes to be seeded, when a 48-well plate is used, the number of cells per well is preferably 10,000 to 100,000 cells, more preferably 20,000 to 80,000 cells, and particularly preferably 30,000 to 60,000 cells.

[0082] In the present invention, after treating cardiomyocytes with a culture medium containing a physiologically active substance of the sympathetic nervous system and / or a physiologically active substance of the parasympathetic nervous system, a drug is brought into contact with the treated cardiomyocytes. For example, the drug (containing a candidate drug substance) can be brought into contact with the cardiomyocytes by adding the drug to the culture medium containing the cardiomyocytes.

[0083] As a drug (including candidate substances for drugs), there is no particular limitation and it can be appropriately selected according to the purpose. For example, compounds such as organic low molecular weight compounds, peptides, proteins, antibodies, nucleic acids, carbohydrates (Carbohydrate), lipids, cell extracts, cell culture supernatants, plant extracts, microbial products, etc. can be mentioned. Compounds, peptides, proteins, and antibodies can also use their own libraries. For example, a compound library prepared using combinatorial chemistry technology can be used. Among them, in the evaluation method of the drug of the present invention, it is possible to evaluate side effects, so it is preferred to use a substance that has been confirmed to have the activity desired as a drug as a drug (candidate substance).

[0084] Drugs (including candidate drugs) include candidate active ingredients in pharmaceuticals across all categories. For example, drugs (including candidate drugs) can be evaluated for therapeutic agents for heart disease. Heart disease refers to arrhythmias or circulatory system diseases associated with arrhythmias. Arrhythmias include tachyarrhythmias and bradyarrhythmias.

[0085] As the drug, for example, there are muscarinic receptor inhibitors such as atropine, tiotropium bromide, glycopyrrolate, and AF-DX116, but the drug is not particularly limited.

[0086] As the drug, for example, there are adrenaline receptor inhibitors, and propranolol, atenolol and the like can be used, but are not particularly limited.

[0087] In the present invention, after the drug is brought into contact with the cardiomyocytes, the effect of the drug on the heart rate of the cardiomyocytes is evaluated.

[0088] To evaluate the effect of a drug on the heart rate of cardiomyocytes, the drug's adrenaline receptor inhibitory effect can be evaluated, or the drug's muscarinic receptor inhibitory effect can be evaluated.

[0089] In the evaluation of the effect of drugs on the heart rate of myocardial cells, for example, based on the measurement results of the extracellular potential of myocardial cells, the interval between the peaks representing the depolarization of the myocardial cells is set as the beating time (seconds), and the beating frequency (heart rate) can be calculated by using the formula: heart rate ( / minute) = 60 (seconds / minute) / beating time (seconds).

[0090] For example, the extracellular potential can be measured by contacting the cardiomyocytes with a multi-electrode array. The multi-electrode array is an electrode in which a plurality of microelectrodes are arranged on a substrate, for example, 1 to 768 are arranged, and 1 to 384 wells for cell culture are arranged, for example, 48 or 96 are arranged. As a multi-electrode array (MEA) system capable of measuring the extracellular potential of cultured cells, for example, a MEA system (manufactured by Axion BioSystems, Inc., Maestro Pro) is commercially available. A multi-electrode array system refers to a system that can simultaneously observe electrical signals from a plurality of cells by a plurality of microelectrodes arranged on a substrate, or that can observe the response of cells to stimulation by applying electrical stimulation to cells or tissues.

[0091] As another example, the beating of cardiomyocytes can be detected by standard electrophysiological methods (eg, patch clamp technique), elastic modulus detection devices (eg, MicroTester (CellScale)), imaging devices, or a combination of these methods and devices.

[0092] When using a multi-well plate with microelectrodes arranged in the wells, the seeding density of cardiomyocytes around the electrodes is preferably 100 to 10,000 cells / mm 2 , more preferably 500 to 8000 cells / mm 2 , especially preferably 1000 to 6000 cells / mm 2. In calculating the seeding density of cardiomyocytes around the electrode, the image obtained as follows can be used: after the cardiomyocytes are seeded on a plate and cultured, they are fixed, nuclear staining is performed, and the electrode and its surroundings are photographed under a microscope. For example, paraformaldehyde or the like can be used to fix the cells. For example, Hoechst33342 or the like can be used for nuclear staining, and the image can be taken under a fluorescence microscope. In the image obtained by photographing the electrode and its surroundings under a fluorescence microscope, the seeding density of the cardiomyocytes around the electrode can be calculated using analysis software.

[0093] According to the present invention, a reagent for performing the method for evaluating a drug of the present invention is provided, comprising an adrenergic receptor agonist and / or a muscarinic receptor agonist. Details of the adrenergic receptor agonist and / or the muscarinic receptor agonist are as described herein.

[0094] According to the present invention, a kit comprising the above-mentioned reagent and culture medium of the present invention is also provided. The kit of the present invention may also contain cardiomyocytes. The details of the culture medium and cardiomyocytes are as described in this specification.

[0095] The present invention will be further specifically described with reference to the following examples, but the present invention is not limited to these examples.

[0096] Example

[0097] Reference Example 1: Method and results for evaluating the effect of adding a muscarinic receptor agonist (carbachol) to serum-free culture medium on heart rate

[0098] Frozen human iPS cell-derived cardiomyocytes (iCell Cardiomyocyte 2.0, manufactured by FUJIFILM Cellular Dynamics, Inc.) were thawed and suspended in thawing medium (iCell Cardiomyocyte Thawing Medium, manufactured by FUJIFILM Cellular Dynamics, Inc.). 50,000 cells were then seeded per well on an MEA plate (CytoView MEA 48, manufactured by Axion BioSystems, Inc.). The seeding density of cardiomyocytes around the electrodes was 2736 cells / mm. 2 After standing for several hours in a CO2 incubator, serum-containing maintenance medium (manufactured by FUJIFILM Cellular Dynamics, Inc., iCell Cardiomyocyte Maintenance Medium) was added and cultured in a CO2 incubator for approximately one week. The day before measurement, the medium was replaced with serum-free medium (manufactured by FUJIFILM Cellular Dynamics, Inc., Serum-free Cardiomyocyte Detection Medium).

[0099] On the day of measurement, an MEA plate containing cardiomyocytes derived from human iPS cells was placed in an MEA system (manufactured by Axion BioSystems, Inc., Maestro Pro). After 20 minutes, the extracellular potential was measured (vehicle control, carbachol concentration 0 μmol / L). Subsequently, 0.01 μmol / L of carbachol (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the MEA plate, and the extracellular potential was measured 20 minutes after the addition. Similarly, carbachol was added in a manner to obtain 0.1 μmol / L, 1 μmol / L, and 10 μmol / L, and the extracellular potential was measured at each concentration 20 minutes after the addition. In addition, the solvent for the carbachol to be added was set to serum-free medium. The cells after measurement were fixed with 4% paraformaldehyde, nuclear staining was performed with Hoechst 33342, and images of the electrodes and their surroundings were taken under a fluorescence microscope (inverted microscope ECLIPSE Ti-U, manufactured by Nikon Corporation). Cardiomyocytes were detected from the acquired images using analysis software (ImageJ, NIH) and normalized by the area of ​​the photographed region to calculate the cardiomyocyte seeding density around the electrodes. In the following experiments, the cardiomyocyte seeding density around the electrodes was also calculated using the same method.

[0100] The results of extracellular potential measurements will show that the intervals between depolarization spikes of human iPS cell-derived cardiomyocytes ( Figure 1 ) is set as the beat time (seconds), and the heart rate ( / min) is calculated as 60 / beat time (seconds). By adding carbachol, it was confirmed that the heart rate ( Figure 2 ).

[0101] Example 1: Method and Results of Evaluating Heart Rate Reduction Induced by a Muscarinic Receptor Agonist (Carbachol) Using Serum-Free Medium Supplemented with an Adrenergic Receptor Agonist (Isoproterenol)

[0102] Frozen human iPS cell-derived cardiomyocytes (iCell Cardiomyocyte 2.0, manufactured by FUJIFILM Cellular Dynamics, Inc.) were thawed and suspended in thawing medium (iCell Cardiomyocyte Thawing Medium, manufactured by FUJIFILM Cellular Dynamics, Inc.). 50,000 cells were then seeded per well on an MEA plate (CytoView MEA 48, manufactured by Axion BioSystems, Inc.). The seeding density of cardiomyocytes around the electrodes was 2671 cells / mm. 2 After standing for several hours in a CO2 incubator, serum-containing maintenance medium (manufactured by FUJIFILM Cellular Dynamics, Inc., iCell Cardiomyocyte Maintenance Medium) was added and cultured in a CO2 incubator for approximately one week. The day before measurement, the medium was replaced with serum-free medium (manufactured by FUJIFILM Cellular Dynamics, Inc., Serum-free Cardiomyocyte Detection Medium).

[0103] On the day of measurement, the MEA plate in which the cardiomyocytes derived from human iPS cells were cultured was set in an MEA system (manufactured by Axion BioSystems, Inc., Maestro Pro). After 1 μmol / L of isoproterenol (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the culture medium, the extracellular potential was measured after 20 minutes from the addition (vehicle control, carbachol concentration 0 μmol / L). Subsequently, 0.01 μmol / L of carbachol (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the MEA plate, and the extracellular potential was measured after 20 minutes from the addition. Similarly, carbachol was added in a manner of 0.1 μmol / L, 1 μmol / L, and 10 μmol / L, and at each concentration, the extracellular potential was measured. The solvent for carbachol to be added was a serum-free medium to which 1 μmol / L isoproterenol had been added so that the isoproterenol concentration in the serum-free medium would not change when carbachol was added.

[0104] Based on the results of extracellular potential measurement, the interval between depolarization spikes of human iPS cell-derived cardiomyocytes was defined as beat time (seconds), and the heart rate ( / min) was calculated as 60 / beat time (seconds). By adding carbachol, it was confirmed that the heart rate decreased by up to 34% ( Figure 3 ).

[0105] Example 2: Method and Results of Evaluating Heart Rate Decrease Induced by Muscarinic Receptor Agonist (Cevimeline) Using Serum-Free Medium Supplemented with Adrenergic Receptor Agonist (Isoproterenol)

[0106] Evaluation was performed using MEA plates cultured with human iPS cell-derived cardiomyocytes in the same manner as in Example 1. Specifically, the same procedures as in Example 1 were performed except that carbachol added in Example 1 was replaced with cevimeline (manufactured by Sigma-Aldrich Co., LLC.) at concentrations of 0.1 μmol / L, 1 μmol / L, 10 μmol / L, and 100 μmol / L.

[0107] Based on the results of extracellular potential measurement, the interval between the depolarization spikes of human iPS cell-derived cardiomyocytes was defined as the beat time (seconds), and the heart rate ( / min) was calculated as 60 / beat time (seconds). By adding cevimeline, a maximum decrease of 26% in heart rate ( Figure 4 )

[0108] Example 3: Method and Results of Evaluating Heart Rate Decrease Induced by a Muscarinic Receptor Agonist (Pilocarpine) Using Serum-Free Medium Supplemented with an Adrenergic Receptor Agonist (Isoproterenol)

[0109] Evaluation was performed using MEA plates cultured with human iPS cell-derived cardiomyocytes in the same manner as in Example 1. Specifically, the same procedures as in Example 1 were performed except that carbachol added in Example 1 was replaced with pilocarpine (manufactured by FUJIFILM Wako Pure Chemical Corporation) at concentrations of 0.1 μmol / L, 1 μmol / L, 10 μmol / L, and 100 μmol / L.

[0110] Based on the results of extracellular potential measurement, the interval between spikes showing depolarization of human iPS cell-derived cardiomyocytes was defined as beat time (seconds), and the heart rate ( / min) was calculated as 60 / beat time (seconds). By adding pilocarpine, it was confirmed that the heart rate decreased by up to 15% ( Figure 5 )

[0111] Comparative Example 1: Method and Results of Evaluating Heart Rate Reduction Induced by Muscarinic Receptor Agonist (Carbachol) Using Maintenance Medium

[0112] Frozen human iPS cell-derived cardiomyocytes (iCell Cardiomyocytes 2.0, manufactured by FUJIFILM Cellular Dynamics, Inc.) were thawed and suspended in thawing medium (iCell Cardiomyocyte Thawing Medium, manufactured by FUJIFILM Cellular Dynamics, Inc.). Then, 50,000 cells were seeded per well on an MEA plate (CytoView MEA48, manufactured by Axion BioSystems, Inc.). The seeding density of cardiomyocytes around the electrodes was 2798 cells / mm. 2 After standing in a CO2 incubator for several hours, serum-containing maintenance medium (iCell Cardiomyocyte Maintenance Medium, manufactured by FUJIFILM Cellular Dynamics, Inc.) was added and cultured in a CO2 incubator for approximately one week. Culture was continued in the maintenance medium until the day of measurement.

[0113] On the day of measurement, the MEA plate in which cardiomyocytes derived from human iPS cells were cultured was placed on a MEA system (manufactured by Axion BioSystems, Inc., Maestro Pro), and after 20 minutes, the extracellular potential was measured (vehicle control, carbachol concentration 0 μmol / L). Subsequently, 0.01 μmol / L of carbachol (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the MEA plate, and after 20 minutes from the start of addition, the extracellular potential was measured. Similarly, carbachol was added in a manner to become 0.1 μmol / L, 1 μmol / L, and 10 μmol / L, and at each concentration, the extracellular potential was measured.

[0114] Based on the results of extracellular potential measurement, the interval between the depolarization spikes of human iPS cell-derived cardiomyocytes was defined as the beat time (seconds), and the heart rate ( / min) was calculated as 60 / beat time (seconds). No significant decrease in heart rate was observed due to the addition of carbachol. Figure 6 ).

[0115] Example 4: Method and Results of Evaluating Heart Rate Increase Induced by Muscarinic Receptor Inhibitor (Atropine) Using Serum-Free Medium Supplemented with an Adrenergic Receptor Agonist (Isoproterenol) and a Muscarinic Receptor Agonist (Carbachol)

[0116] Frozen human iPS cell-derived cardiomyocytes (iCell Cardiomyocytes 2.0, manufactured by FUJIFILM Cellular Dynamics, Inc.) were thawed and suspended in thawing medium (iCell Cardiomyocyte Thawing Medium, manufactured by FUJIFILM Cellular Dynamics, Inc.). 50,000 cells were then seeded per well on an MEA plate (CytoView MEA48, manufactured by Axion BioSystems, Inc.). The seeding density of cardiomyocytes around the electrodes was 2725 cells / mm 2 After standing for several hours in a CO2 incubator, serum-containing maintenance medium (manufactured by FUJIFILM Cellular Dynamics, Inc., iCell Cardiomyocyte Maintenance Medium) was added and cultured in a CO2 incubator for approximately one week. The day before measurement, the medium was replaced with serum-free medium (manufactured by FUJIFILM Cellular Dynamics, Inc., Serum-free Cardiomyocyte Detection Medium).

[0117] On the day of measurement, the MEA plate in which the cardiomyocytes derived from human iPS cells were cultured was set in the MEA system (manufactured by Axion BioSystems, Inc., Maestro Pro), and 1 μmol / L of isoproterenol (manufactured by Tokyo Chemical Industry Co., Ltd.) and 3 μmol / L of carbachol (manufactured by Tokyo Chemical Industry Co., Ltd.) were added to the culture medium. After 20 minutes from the start of addition, the extracellular potential was measured (vehicle control, atropine concentration 0 μmol / L). Subsequently, 0.01 μmol / L of atropine (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the MEA plate, and after 20 minutes from the start of addition, the extracellular potential was measured. Similarly, atropine was added in a manner of 0.1 μmol / L and 1 μmol / L, and at each concentration, the extracellular potential was measured. The solvent for atropine to be added was a serum-free medium containing 1 μmol / L isoproterenol and 3 μmol / L carbachol so that the concentrations of isoproterenol and carbachol in the serum-free medium would not change when atropine was added.

[0118] Based on the results of extracellular potential measurement, the interval between the depolarization spikes of human iPS cell-derived cardiomyocytes was defined as the beat time (seconds), and the heart rate ( / min) was calculated as 60 / beat time (seconds). The addition of atropine was found to increase the heart rate by up to 51%. Figure 7 ).

[0119] Example 5: Method and Results of Evaluating Heart Rate Increase Induced by Muscarinic Receptor Inhibitor (Tiotropium) Using Serum-Free Medium Supplemented with Adrenergic Receptor Agonist (Isoproterenol) and Muscarinic Receptor Agonist (Carbachol)

[0120] Evaluation was performed using MEA plates cultured with human iPS cell-derived cardiomyocytes in the same manner as in Example 4. Specifically, the same procedures as in Example 4 were performed except that the atropine added in Example 4 was replaced with tiotropium bromide (manufactured by Tokyo Chemical Industry Co., Ltd.) at concentrations of 1 pmol / L, 10 pmol / L, 100 pmol / L, and 1000 pmol / L.

[0121] Based on the results of extracellular potential measurement, the interval between spikes showing depolarization of human iPS cell-derived cardiomyocytes was defined as beat time (seconds), and the heart rate ( / min) was calculated as 60 / beat time (seconds). The heart rate was confirmed to increase in a concentration-dependent manner by the addition of tiotropium bromide, with a maximum increase of 36% ( Figure 8 ).

[0122] Example 6: Method and Results of Evaluating Heart Rate Increase Induced by Muscarinic Receptor Inhibitor (Glycopyrrolate) Using Serum-Free Medium Supplemented with an Adrenergic Receptor Agonist (Isoproterenol) and a Muscarinic Receptor Agonist (Carbachol)

[0123] Evaluation was performed using MEA plates cultured with human iPS cell-derived cardiomyocytes in the same manner as in Example 4. Specifically, the same procedures as in Example 4 were performed except that the atropine added in Example 4 was replaced with glycopyrrolate (manufactured by Tokyo Chemical Industry Co., Ltd.) at concentrations of 0.01 nmol / L, 0.1 nmol / L, 1 nmol / L, and 10 nmol / L.

[0124] Based on the results of extracellular potential measurement, the interval between spikes showing depolarization of cardiomyocytes derived from human iPS cells was defined as beat time (seconds), and the heart rate ( / min) was calculated as 60 / beat time (seconds). By adding glycopyrrolate, it was confirmed that the heart rate increased in a concentration-dependent manner, and the maximum increase in heart rate was 36% ( Figure 9 ).

[0125] Example 7: Method and results for evaluating the effect of adding an adrenergic receptor inhibitor (propranolol) on heart rate using serum-free culture medium supplemented with an adrenergic receptor agonist (isoproterenol) and a muscarinic receptor agonist (carbachol)

[0126] Evaluation was performed using MEA plates cultured with human iPS cell-derived cardiomyocytes in the same manner as in Example 4. Specifically, the same procedures as in Example 4 were performed except that the atropine added in Example 4 was replaced with propranolol (manufactured by FUJIFILM Wako Pure Chemical Corporation) at propranolol concentrations of 0.1 nmol / L, 1 nmol / L, 10 nmol / L, and 100 nmol / L.

[0127] Based on the results of extracellular potential measurement, the interval between spikes showing depolarization of human iPS cell-derived cardiomyocytes was defined as beat time (seconds), and the heart rate ( / min) was calculated as 60 / beat time (seconds). It was confirmed that the heart rate decreased in a concentration-dependent manner by adding propranolol, and a maximum decrease of 28% in heart rate ( Figure 10 ).

[0128] Example 8: Method and results for evaluating the effect of the addition of an adrenergic receptor inhibitor (atenolol) on heart rate using serum-free culture medium supplemented with an adrenergic receptor agonist (isoproterenol) and a muscarinic receptor agonist (carbachol)

[0129] Evaluation was performed using MEA plates cultured with human iPS cell-derived cardiomyocytes in the same manner as in Example 4. Specifically, the same procedures as in Example 4 were performed except that atenolol (manufactured by Tokyo Chemical Industry Co., Ltd.) was used instead of atropine in Example 4, and the concentrations of atenolol added were 0.01 μmol / L, 0.1 μmol / L, 1 μmol / L, and 10 μmol / L.

[0130] Based on the results of extracellular potential measurement, the interval between spikes showing depolarization of human iPS cell-derived cardiomyocytes was defined as beat time (seconds), and the heart rate ( / min) was calculated as 60 / beat time (seconds). It was confirmed that the heart rate decreased in a concentration-dependent manner by the addition of atenolol, and a maximum decrease of 22% in heart rate ( Figure 11 ).

[0131] Example 9: Method and results for evaluating the effects of adding a drug (aspirin) that has no effect on heart rate using serum-free culture medium supplemented with an adrenergic receptor agonist (isoproterenol) and a muscarinic receptor agonist (carbachol)

[0132] Frozen human iPS cell-derived cardiomyocytes (iCell Cardiomyocyte 2.0, manufactured by FUJIFILM Cellular Dynamics, Inc.) were thawed and suspended in thawing medium (iCell Cardiomyocyte Thawing Medium, manufactured by FUJIFILM Cellular Dynamics, Inc.). 50,000 cells were then seeded per well on an MEA plate (CytoView MEA48, manufactured by Axion BioSystems, Inc.). The seeding density of cardiomyocytes around the electrodes was 2783 cells / mm. 2 After standing for several hours in a CO2 incubator, serum-containing maintenance medium (manufactured by FUJIFILM Cellular Dynamics, Inc., iCell Cardiomyocyte Maintenance Medium) was added and cultured in a CO2 incubator for approximately one week. The day before measurement, the medium was replaced with serum-free medium (manufactured by FUJIFILM Cellular Dynamics, Inc., Serum-free Cardiomyocyte Detection Medium).

[0133] On the day of measurement, the MEA plate in which the cardiomyocytes derived from human iPS cells were cultured was set in an MEA system (manufactured by Axion BioSystems, Inc., Maestro Pro). After 1 μmol / L of isoproterenol (manufactured by Tokyo Chemical Industry Co., Ltd.) and 3 μmol / L of carbachol (manufactured by Tokyo Chemical Industry Co., Ltd.) were added to the culture medium, the extracellular potential (vehicle control, aspirin concentration 0 μmol / L) was measured after 20 minutes from the start of addition. Subsequently, 0.1 μmol / L of aspirin (manufactured by FUJIFILM Wako Pure Chemical Corporation) was added to the MEA plate, and the extracellular potential was measured after 20 minutes from the start of addition. Similarly, aspirin was added in a manner of 1 μmol / L and 10 μmol / L, and at each concentration, the extracellular potential was measured. The solvent for aspirin to be added was a serum-free medium containing 1 μmol / L isoproterenol and 3 μmol / L carbachol so that the concentrations of isoproterenol and carbachol in the serum-free medium would not change when aspirin was added.

[0134] Based on the results of extracellular potential measurement, the interval between spikes showing depolarization of human iPS cell-derived cardiomyocytes was defined as beat time (seconds), and the heart rate ( / min) was calculated as 60 / beat time (seconds). The maximum increase in heart rate due to the addition of aspirin was 17% ( Figure 12 ).

[0135] Comparative Example 3: Method and Results of Evaluating Heart Rate Increase Induced by Muscarinic Receptor Inhibitor (Atropine) Using Maintenance Medium

[0136] Frozen human iPS cell-derived cardiomyocytes (iCell Cardiomyocytes 2.0, manufactured by FUJIFILM Cellular Dynamics, Inc.) were thawed and suspended in thawing medium (iCell Cardiomyocyte Thawing Medium, manufactured by FUJIFILM Cellular Dynamics, Inc.). 50,000 cells were then seeded per well on an MEA plate (CytoView MEA48, manufactured by Axion BioSystems, Inc.). The seeding density of cardiomyocytes around the electrodes was 2662 cells / mm. 2After standing in a CO2 incubator for several hours, serum-containing maintenance medium (iCell Cardiomyocyte Maintenance Medium, manufactured by FUJIFILM Cellular Dynamics, Inc.) was added and cultured in a CO2 incubator for approximately one week. Culture was continued in the maintenance medium until the day of measurement.

[0137] On the day of measurement, an MEA plate containing cardiomyocytes derived from human iPS cells was placed on an MEA system (Axion BioSystems, Inc., Maestro Pro). After 20 minutes, the extracellular potential was measured (vehicle control, atropine concentration 0 μmol / L). Subsequently, 0.01 μmol / L of atropine (Tokyo Chemical Industry Co., Ltd.) was added to the MEA plate, and 20 minutes after the addition, the extracellular potential was measured. Similarly, atropine was added to 0.1 μmol / L and 1 μmol / L, and the extracellular potential was measured at each concentration.

[0138] Based on the results of extracellular potential measurement, the interval between spikes showing depolarization of human iPS cell-derived cardiomyocytes was defined as the beat time (seconds), and the heart rate ( / min) was calculated as 60 / beat time (seconds). No significant increase in heart rate was observed after the addition of atropine. Figure 13 ).

[0139] Comparative Example 4: Method and Results of Evaluating Heart Rate Increase Induced by Muscarinic Receptor Inhibitor (Atropine) Using Serum-Free Medium

[0140] Frozen human iPS cell-derived cardiomyocytes (iCell Cardiomyocytes 2.0, manufactured by FUJIFILM Cellular Dynamics, Inc.) were thawed and suspended in thawing medium (iCell Cardiomyocyte Thawing Medium, manufactured by FUJIFILM Cellular Dynamics, Inc.). 50,000 cells were then seeded per well on an MEA plate (CytoView MEA48, manufactured by Axion BioSystems, Inc.). The seeding density of cardiomyocytes around the electrodes was 2703 cells / mm 2After standing for several hours in a CO2 incubator, serum-containing maintenance medium (manufactured by FUJIFILM Cellular Dynamics, Inc., iCell Cardiomyocyte Maintenance Medium) was added and cultured in a CO2 incubator for approximately one week. The day before measurement, the medium was replaced with serum-free medium (manufactured by FUJIFILM Cellular Dynamics, Inc., Serum-free Cardiomyocyte Detection Medium).

[0141] On the day of measurement, the MEA plate in which cardiomyocytes derived from human iPS cells were cultured was placed on a MEA system (manufactured by Axion BioSystems, Inc., Maestro Pro), and after 20 minutes, the extracellular potential was measured (vehicle control, atropine concentration 0 μmol / L). Subsequently, 0.01 μmol / L of atropine (manufactured by Tokyo Chemical Industry Co., Ltd.) was added to the MEA plate, and after 20 minutes from the start of addition, the extracellular potential was measured. Similarly, atropine was added in a manner to become 0.1 μmol / L and 1 μmol / L, and after 20 minutes from the start of addition, the extracellular potential was measured at each concentration.

[0142] Based on the results of extracellular potential measurement, the interval between spikes showing depolarization of human iPS cell-derived cardiomyocytes was defined as the beat time (seconds), and the heart rate ( / min) was calculated as 60 (seconds / min) / beat time (seconds). No significant increase in heart rate was observed after the addition of atropine. Figure 14 ).

Claims

1. A method for evaluating a drug, comprising the following steps: a step of treating cardiomyocytes with a culture medium containing a physiologically active substance of the sympathetic nervous system and / or a physiologically active substance of the parasympathetic nervous system; a step of contacting a drug with the treated cardiomyocytes; and Steps for evaluating the effects of drugs on the heart rate of cardiomyocytes.

2. The method according to claim 1, wherein The culture medium is serum-free.

3. The method according to claim 1 or 2, wherein: The physiologically active substance of the sympathetic nervous system includes an agonist of adrenaline receptors.

4. The method according to claim 1 or 2, wherein: The physiologically active substance of the parasympathetic nervous system includes an agonist of a muscarinic receptor.

5. The method according to claim 1 or 2, comprising the steps of: The inhibitory effect of the drug on adrenaline receptors was evaluated.

6. The method according to claim 1 or 2, comprising the steps of: The inhibitory effects of the drugs on muscarinic receptors were evaluated.

7. The method according to claim 1 or 2, wherein: The cardiomyocytes are cardiomyocytes derived from human iPS cells.

8. The method according to claim 1 or 2, wherein: The physiologically active substance of the parasympathetic nervous system is carbachol, and the physiologically active substance of the sympathetic nervous system is isoproterenol.

9. A reagent for performing the method according to claim 1 or 2, wherein the reagent comprises an adrenergic receptor agonist and / or a muscarinic receptor agonist.

10. A kit comprising the reagent according to claim 9 and a culture medium. The kit according to claim 10 , further comprising cardiomyocytes.

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