JP2020071206A - Method for determining cardiac toxicity based on atp active measurement - Google Patents

Abstract

To provide a new method for determining whether a drug will cause a drug-induced long QT syndrome in the field of drug development instead of a cell membrane potential measuring method.SOLUTION: There is provided a method for determining whether an inspection target material will cause a cardiac toxicity, and the method includes the steps of: injecting an inspection target material into a nonhuman transgenic animal which presents a fluorescent protein containing an ATP bonding part, a fluorescent protein as a donor for transferring a fluorescent resonance energy, and a fluorescent protein as an acceptor; measuring a fluorescent signal from the heart of the nonhuman transgenic animal; and determining whether the inspection target material will cause a cardiac toxicity according to the measured fluorescence signal.SELECTED DRAWING: None

Description

  The present invention relates to a method for determining whether a test substance causes cardiotoxicity. The present invention also relates to a method for producing a drug candidate substance library, and a cardiomyocyte or non-human transgenic animal for determining whether a test substance causes cardiotoxicity.

  In drug development, it is important to properly evaluate its efficacy and toxicity. As a drug toxicity, drug-induced long QT syndrome that causes serious arrhythmia in a patient is known. In the drug-induced long QT syndrome, the QT interval on the electrocardiogram is prolonged after administration of the drug, and non-sustained polymorphic ventricular tachycardia (Torsade de Pointes; TdP) often causes ventricular fibrillation, resulting in syncope. Or, it is a serious condition that causes sudden death.

  Prolongation of QT interval in drug-induced prolongation of QT syndrome is considered to be caused by a drug administered with a harmonious heart beat, which acts on the ion channels of cardiomyocytes and disturbs its harmonies. A cell membrane potential measurement method is known as a method for determining whether a drug causes drug-induced long QT syndrome (Non-patent Document 1).

Pharmacological magazine (2005) 126, p321-327.

  Drugs such as Verapamil and Vanoxerine are known to act on the ion channel hERG channel but not to prolong the QT interval. Therefore, there is a need in the field of drug development for new alternatives to cell membrane potential measurement in determining whether a drug causes drug-induced long QT syndrome.

  In the development of pharmaceuticals, candidate substances for which arrhythmia was observed as a side effect during the development process are excluded from the development process and are not marketed. On the other hand, in the development process, some arrhythmias were not seen as a side effect, and some of the drugs that were launched on the market were those that caused arrhythmia in the administered patients. If an arrhythmia as a side effect was observed after launch, and if an arrhythmia as a side effect was observed in the late stage of development even before launch, there was a problem of wasting the development cost of the drug until then. .. Therefore, in the field of drug development, there is a demand for a method capable of determining whether or not a drug candidate substance causes arrhythmia earlier.

  The present inventor has found that there is a change in the ATP concentration in the heart even in the presence of a drug at a low concentration that does not show signs such as QT prolongation on the electrocardiogram. Thus, the present inventor has found that it is possible to determine whether or not the test substance causes cardiotoxicity such as QT prolongation syndrome by measuring the ATP concentration in the heart, and completed the present invention. That is, the present invention relates to the following.

[Claim 1] A method for determining whether or not a test substance causes cardiotoxicity, the test substance comprising an ATP-binding moiety, a fluorescent protein as a donor for fluorescence resonance energy transfer, and a fluorescent protein as an acceptor. Administering to a non-human transgenic animal expressing a fusion protein comprising a fusion protein comprising: a step of measuring a fluorescence signal from the heart of the non-human transgenic animal; and, based on the measured fluorescence signal, A determination method comprising a step of determining whether or not a test substance causes cardiotoxicity.

[Claim 2] The non-human transgenic animal contains DNA encoding the fusion protein inserted into the ROSA26 locus on its chromosome, and the DNA is operably linked to a CAG promoter sequence, The determination method according to Item 1.
[Claim 3] A method for determining whether or not a test substance causes cardiotoxicity, the test substance comprising an ATP-binding moiety, a fluorescent protein as a donor for fluorescence resonance energy transfer, and a fluorescent protein as an acceptor. A step of contacting a cardiomyocyte expressing a fusion protein containing a fusion protein containing; a step of measuring a fluorescent signal from the cardiomyocyte; and whether the test substance causes cardiotoxicity based on the measured fluorescent signal A determination method including a step of determining whether or not.
[Item 4] In item 3, wherein the cardiomyocyte contains DNA encoding the fusion protein inserted into the ROSA26 gene locus on its chromosome, and the DNA is operably linked to a CAG promoter sequence. Judgment method described.

[Item 5] The determination method according to any one of Items 1 to 4, wherein the determination step includes a step of comparing the fluorescence signal with a first threshold value.
[Claim 6] The ATP-binding portion is a CBS domain of inosine monophosphate dehydrogenase 2 (IMPDH2) or a fragment thereof or a variant thereof, GlnK1 or a fragment thereof or a variant thereof, or an ε subunit of ATP synthase or a portion thereof. Item 6. The determination method according to any one of Items 1 to 5, which is a fragment or a modified form thereof.
[Item 7] The determination method according to any one of Items 1 to 6, wherein the ATP-binding portion is an ε subunit of ATP synthase, a fragment thereof, or a modified form thereof.
[Item 8] In the fusion protein, one of the fluorescent protein as the donor and the fluorescent protein as the acceptor is linked to the amino terminal side of the ATP-binding portion, and the other is carboxy of the ATP-binding portion. Item 8. The determination method according to any one of Items 1 to 7, which is linked to the terminal side.

[Item 9] The fluorescence signal is derived from a fluorescent protein serving as a donor (hereinafter referred to as “donor side fluorescent intensity”), and a fluorescent intensity derived from a fluorescent protein serving as an acceptor (hereinafter referred to as “acceptor side fluorescent intensity”). 1), the ratio of the fluorescence intensity on the acceptor side to the fluorescence intensity on the donor side (= [fluorescence intensity on acceptor side] / [fluorescence intensity on donor side]), or the fluorescence lifetime of the fluorescent protein as a donor. Item 9. The determination method according to any one of Item 8.
[Item 10] The determination method according to any one of Items 1 to 9, wherein the non-human transgenic animal is a transgenic mouse.
[Item 11] Any one of Items 3 to 9, wherein the cardiomyocytes are non-human transgenic animal-derived cardiomyocytes expressing the fusion protein or cardiomyocytes prepared from stem cells expressing the fusion protein. The determination method according to one item.

[Claim 12] A method for producing a drug candidate substance library, wherein the determination is made as to whether or not each of two or more test substances causes cardiotoxicity. And a step of producing a library of drug candidate substances by selecting a test substance which has been determined not to cause cardiotoxicity in the process.
[Claim 13] A cardiomyocyte comprising an ATP-binding moiety for determining whether a test substance causes cardiotoxicity, a fluorescent protein as a donor for fluorescence resonance energy transfer, and a fluorescent protein as an acceptor, or Non-human transgenic animals expressing fusion proteins, including fusion proteins.
[Claim 14] A method for determining whether or not a test substance causes cardiotoxicity, comprising the steps of contacting the test substance with cardiomyocytes, and measuring the ATP concentration in the cardiomyocytes; or The method further comprises the step of administering to an animal, and the step of measuring the ATP concentration in the heart of the non-human animal, and further the step of determining whether or not the test substance causes cardiotoxicity based on the measured ATP concentration. , Judgment method.

A conceptual diagram when the supply and demand of ATP in the heart are balanced (Fig. 1a), a conceptual diagram when the supply of ATP is smaller than the demand of ATP (Fig. 1b), and ATP for the demand of ATP Schematic diagram when the supply of water is large (Fig. 1c). Fluorescence images of hearts of transgenic mice administered with physiological saline (Fig. 2a: 0 min after administration, Fig. 2b: 60 min after administration). An electrocardiogram at 0 minutes after the start of administration of a saline solution (FIG. 2c), and an electrocardiogram at 60 minutes after the start of administration (FIG. 2d). The graph which shows the ATP density | concentration change in the heart of the said transgenic mouse (FIG. 2e). Electrocardiograms of transgenic mice before and after the start of disopyramide administration (before the start of administration: on top of FIG. 3a, after the start of administration: bottom of FIG. 3a). A fluorescence image of the heart of the transgenic mouse (start of administration 0 min: left of FIG. 3b, post-administration 60 min: right of FIG. 3b), and a graph showing changes in ATP concentration in the heart (FIG. 3c). Nifekalant (Fig. 4a), Levofloxacin (Fig. 4b), Alfuzosin hydrochloride (Fig. 4c), Procainamide (Fig. 4d), Droperidol (Fig. 4e), and Erythromycin (Fig. 4f) were administered to the hearts of transgenic mice (Fig. 4f). 0 min after start: upper left figure, 60 min after start of administration: upper right figure) and a graph showing changes in ATP concentration (lower figure). Fluorescence image of the heart of a transgenic mouse administered with Amphotericin B (Fig. 5a), Azithromycin (Fig. 5b), Ciprofloxacin (Fig. 5c), and Metronidazole (Fig. 5d) (0 min after administration: upper left, 60 min after administration: (Upper right figure) and a graph showing changes in ATP concentration (lower figure). Fluorescence images of the hearts of transgenic mice to which Vanoxerine (Fig. 6a), Verapamil (Fig. 6b) and Furosemide (Fig. 6c) were administered (0min after administration: upper left, 60min after administration: upper right) and ATP concentration in the heart. Graph showing changes (lower figure). Fluorescence images of the heart of transgenic mice administered with 5-FU (Fig. 7a), Cyclophosphamide (Fig. 7b), and Ifosfamide (Fig. 7c) (0 min after administration: upper left, 60 min after administration: upper right) and heart. 3 is a graph showing changes in ATP concentration in FIG.

  As used herein, "cardiotoxicity" means drug-induced toxicity that adversely affects the heart. Cardiotoxicity includes, but is not limited to, arrhythmias, heart failure, and long QT syndrome.

  As used herein, the term “arrhythmia” refers to a state in which the rhythm function of the cardiac functions is abnormal. Arrhythmias include, but are not limited to, arrhythmias, supraventricular tachycardia, "ventricular extrasystole", "atrial (supraventricular) extrasystole", "sustained ventricular tachycardia", "atrial fibrillation". , "Atrial tachycardia", "atrial flutter", "atrioventricular block" and "sinus insufficiency syndrome". In the present specification, “proarrhythmia” means that existing arrhythmia is exacerbated or new arrhythmia occurs by administration of a substance. As used herein, “supraventricular tachycardia” means a tachycardia in which the source of stimulation is at the sinus node, atrial muscle or ventricular junction.

  In the present specification, “extra systole” means a state in which, apart from the regular beats generated by the sinus node, excitement starts from another site and the heart contracts faster than the stimulation from the sinus node. As used herein, "ventricular extrasystole" means extrasystole that occurs in the ventricle below the atrioventricular node where the stimulus occurs. Premature ventricular contractions can be induced by, but not limited to, sympathomimetic drugs such as catecholamines or caffeine.

  As used herein, the term "atrial (supraventricular) premature contraction" means premature contraction from the atrium to the ventricular nodule where the stimulus occurs. Premature atrial (supraventricular) contractions can be induced by bronchodilators, such as theophylline, anticholinergics or β2-agonists. When the state of supraventricular extrasystole worsens, "atrial fibrillation", "atrial tachycardia" or "atrial flutter" may develop. As used herein, “atrial fibrillation” means a state in which the atrial heart rate becomes completely irregular and high frequency. As used herein, "atrial tachycardia" means paroxysmal tachycardia of atrial origin. In the present specification, “atrial flutter” means a state in which the atria do not excite and contract in response to a stimulus from a sinus node, but continue the regular and frequent excitement occurring in the atrium itself.

  As used herein, "atrioventricular block" means impaired conduction of conduction between the atria and ventricles. As used herein, "sinus dysfunction syndrome" means a condition that causes bradycardia due to the sinus node or the conduction system downstream of the sinus node.

  In the present specification, "heart failure" is mainly caused by shortness of breath due to impaired perfusion in various organs in the body and peripheral limbs due to dysfunction of the heart (mainly left heart), and causes palpitation, malaise, decrease in exercise tolerance, etc. Means a syndrome. Heart failure includes, but is not limited to, left heart failure, right heart failure and bilateral heart failure. In one example, the heart failure is left heart failure or bilateral heart failure.

  As used herein, “long QT syndrome (LQTS)” means drug-induced long QT syndrome, which causes severe ventricular arrhythmias (eg, “ventricular tachycardia” and “ventricular fibrillation”), dizziness, cerebral ischemia. By a syndrome that can cause symptoms (eg syncope) or sudden death. As used herein, "ventricular tachycardia (VT)" refers to paroxysmal tachycardia that occurs at the ectopic origin of the ventricles. In the present specification, “ventricular fibrillation (VF)” means a state in which individual ventricular proper muscles are excited in a chaotic manner, irrespective of the excitation of the ventricles from the upper centers. As used herein, "torsade de pointe (TdP)" means transient ventricular fibrillation or polymorphic ventricular tachycardia.

  In the present specification, the “test substance” includes, but is not limited to, a substance that is interested in use as a drug, a substance developed as a drug candidate substance, or a substance used as a drug. The test substance is, for example, an organic molecule, a nucleic acid, a protein, or a lipid, or a combination thereof. The test substance can be prepared by a known method, or is commercially available.

  In one example, the test substance is administered to the animal according to known methods. Administration of the test substance to the animal is, but not limited to, oral administration or parenteral administration. In another example, the test substance is contacted with the cells according to known methods. The contact of the test substance with the cells is, for example, when the cells are cultured, but is not limited thereto, and the test substance may be added to the cell culture medium. The test substance may be, for example, one kind or a combination of two or more kinds of test substances.

  In the present specification, “two or more test substances” used in the production of a library of drug candidate substances include, but are not limited to, 10 or more, 100 or more, or 1000 or more test substances. Including. The two or more test substances used in the production of the drug candidate substance library are, for example, compound libraries (URL: http://www.cbrg.riken.jp/npd/ja/ provided by RIKEN. Announce.html), various compound libraries (https://www.ddi.u-tokyo.ac.jp/chemical_library/) managed or provided by the University of Tokyo Drug Discovery Agency, commercially available from Funakoshi Co., Ltd. A compound library (TOCRISCREEN (registered trademark)), and various compound libraries commercially available from Sigma-Aldrich Japan GK (for example, SALOR) are included.

  As used herein, "fusion protein" means a protein containing a fluorescent protein as a donor for fluorescence resonance energy transfer (FRET), a fluorescent protein as an acceptor, and an ATP binding moiety. The fusion protein includes, but is not limited to, a DNA encoding a gene for a known ATP-binding portion, a DNA encoding a fluorescent protein as a FRET donor, and a DNA encoding a fluorescent protein as an acceptor. It can be prepared by genetic engineering technology. In one example, the fusion protein can be prepared according to the method described in Nakano, M, ACS Chem Biol, 2011; 6,709-715.

  The fusion protein is not limited, but either one of the fluorescent protein as a donor for FRET and the fluorescent protein as an acceptor is linked to the amino-terminal (A-terminal) side of the ATP-binding portion. The other fluorescent protein is linked to the carboxy terminal (C terminal) side of the ATP binding portion. In one example, one of the two types of fluorescent proteins for FRET is linked to the A-terminal side of the ATP binding part, and the other of the two types of fluorescent proteins is linked to the C-terminal side of the ATP binding part. There is. The two types of fluorescent proteins may be linked to the ATP binding part via a linker sequence.

  The two types of fluorescent proteins are, but are not limited to, arranged so as to cause FRET when ATP binds to the ATP binding portion of the fusion protein. In another example, the two types of fluorescent proteins are arranged so that FRET is eliminated when ATP binds to the ATP-binding portion of the fusion protein.

  In the present specification, "fluorescence resonance energy transfer (FRET)" means that when a fluorescent protein as a donor and a fluorescent protein as an acceptor come within a certain distance, the light energy absorbed by the fluorescent protein as a donor becomes as an acceptor. It means the phenomenon of migration to fluorescent proteins. Detection of FRET is usually performed by irradiating a fluorescent protein as a donor with an excitation wavelength and measuring a fluorescent signal derived from the fluorescent protein as a donor or an acceptor.

  In one example, when measuring the fluorescence intensity derived from a fluorescent protein as a donor, if two types of fluorescent proteins are located at a position closer than a certain distance, the fluorescent protein as an acceptor absorbs light energy, so that The fluorescence intensity derived from the fluorescent protein decreases, and the fluorescence intensity increases when the distance is greater than a certain distance. Conversely, when measuring the fluorescence intensity derived from the fluorescent protein as the acceptor, the fluorescence intensity derived from the fluorescent protein as the acceptor increases when the distance is closer than a certain distance, and when the distance is greater than the certain distance. The fluorescence intensity becomes smaller.

  In another example, when measuring the fluorescence lifetime derived from a fluorescent protein as a donor, if the fluorescent protein as a donor and the fluorescent protein as an acceptor are located at a position distant from a certain distance, the fluorescent protein as a donor When they show intrinsic fluorescence lifetimes and their distance is closer than a certain distance, the fluorescence lifetime of the fluorescent protein as a donor becomes short.

  As used herein, “fluorescent protein” includes, but is not limited to, cyan fluorescent protein (CFP), yellow fluorescent protein (YFP), blue fluorescent protein (BFP), green fluorescent protein ( GFP), Red Fluorescent Protein (RFP) and Orange Fluorescent Protein (OFP). In another example, Nature Methods vol. 9, No. The fluorescent protein described in Table 1 of 10,1005-1012 (2012) can also be used. Fluorescent proteins can be prepared by known methods or are commercially available. For example, CFP is Invitrogen CFP, YFP is Invitrogen YFP or Evrogen Phi-Yellow, GFP is Clontech EGFP or Evrogen Tag-GFP, and RFP is Clontech. DsRed2-monomer or HcRed-Tandem from Evrogen.

  The two types of fluorescent proteins for FRET in the fusion protein include, but are not limited to, two types of FRET-producing fluorescent proteins selected from the group consisting of CFP, YFP, BFP, GFP, RFP and OFP. Is a combination of. In one example, the combination of two fluorescent proteins that produce FRET is selected from the group consisting of FRP and OFP, with one fluorescent protein selected from the group consisting of CFP, YFT, BFP and GFP, and 1 A combination with a fluorescent protein as an acceptor of the species. In one example, the combination of two fluorescent proteins that produce FRET includes a combination of CFP and YFP, a combination of BFP and GFP, or a combination of GFP and RFP. Either a fluorescent protein as a donor or a fluorescent protein as an acceptor may be linked to the A-terminal side or the C-terminal side of the ATP-binding portion.

  In the present specification, the “ATP-binding moiety” means a peptide that undergoes a structural change in its three-dimensional structure upon binding or dissociation with adenosine triphosphate (ATP). The ATP-binding portion is not limited, but the binding with adenosine diphosphate (ADP), adenosine monophosphate (AMP), or adenosine acid is 10 times or more than that with ATP. Double or more, or 1000 or more times smaller. In one example, the ATP binding moiety is the CBS domain of inosine monophosphate dehydrogenase 2 (IMPDH2). In this example, a fusion protein containing the CBS domain and CFP and YFP as donors and acceptors for FRET is known (Nat Biotechnol. 2007; 25 (2): 170-172.).

  In another example, the ATP-binding moiety includes GlnK1 or a fragment or variant thereof. GlnK1 is a trimeric PII-like signaling protein known to be involved in the regulation of ammonium assimilation and nitrogen fixation in bacteria. In this example, a fusion protein containing the above Glnk1 and CFP and YFP (circularly permuted monomeric Venus) as donors and acceptors for FRET is known (https://www.ncbi.nlm.nih.gov/). pmc / articles / PMC263334 /).

In another example, the ATP binding moiety includes the ε subunit of ATP synthase F ₀ F ₁ ATPase, or a fragment or variant thereof. The ε subunit may be derived from any species. The ATP binding moiety is, but is not limited to, the ε subunit of microbial origin. ATP-binding moieties are described, for example, in Bacillus sp. It is an ε subunit derived from PS3 (GenBank accession No. AB044942) or from Bacillus subtilis (GenBank accession No. Z28592).

  The ε subunit contains two domains, an N-terminal domain and a C-terminal domain. The N-terminal domain consists of about 85 amino acids from the N-terminal side of the ε subunit, and consists of about 10 β strands. The C-terminal domain is composed of about 45 amino acids from the C-terminal side of the ε subunit, and consists of two α-helices. The C-terminal domain shows a folded crystal structure when the ε subunit alone. The ε subunit shows an extended structure when it forms a complex with the γ subunit which is another subunit of ATP synthase. In addition, from the results of biochemical experiments, in the complex with the γ subunit and the like, the C-terminal domain of the ε subunit undergoes a structural change depending on the presence or absence of ATP, and if there is no ATP, it is in an extended state and ATP exists. Then it is shown that it has a folded structure. From these, it is considered that the C-terminal domain of the ε subunit is extended or easily moved in the absence of ATP, and becomes a folded and compact state when ATP binds.

  The ε subunit may be, for example, full length, a fragment or a variant thereof. The fragment may be a peptide that provides a structure responsible for at least a site capable of causing a structural change in the ε protein in an ATP-specific manner. In one example, the fragment is a peptide containing two domains, an N-terminal domain and a C-terminal domain, which can undergo a structural change due to the presence of ATP. In another example, the variant is not limited to the amino acid sequence of a natural type ε subunit or a fragment thereof, but one or several (for example, 2 to 5) amino acids are substituted or deleted. , Inserted or added. In one example, in order to prevent a substance capable of inhibiting the structural change of the ε protein due to the reaction of the ε subunit and ATP from binding to the ε subunit, the amino acid sequence of the full length of the ε subunit or a fragment thereof is partially substituted. You may. In another example, in the ε subunit or a fragment thereof, a hydrophobic amino acid residue required for interaction with another subunit, γ subunit, can be replaced with a hydrophilic amino acid residue. ..

  As the ATP binding portion, an ε subunit obtained from a bacterial species other than the above bacterial species can also be used. It is known that the ε subunit has different affinities for ATP in different species (J. Biological Chemistry 2003; 278, 36013-36016, and FEBS Letters 2005; 579, 6875-6878). As the fusion protein, various fusion proteins can be prepared from those in which the fluorescence signal by FRET changes at low ATP concentration to those in which the fluorescence signal by FRET changes at high ATP concentration depending on the ATP binding moiety. it can.

  The reactivity of the fusion protein with respect to the ATP concentration can be modified by introducing a mutation into the ATP-binding portion in addition to the biological species of the ATP-binding portion. The DNA encoding the ATP binding part can be prepared by a known method. Mutagenesis in the DNA encoding the ATP-binding portion can be performed according to a known method. The modified ATP-binding moiety having desired properties is not limited to, but is produced by producing a modified ATP-binding moiety from mutated DNA and screening the modified product according to a screening method using an ELISA or a phage display method. ,Obtainable. The obtained modified ATP-binding portion can be measured for its binding constant or dissociation constant using surface plasmon resonance, but is not limited thereto. Based on the measured binding constant or dissociation constant, a fusion protein in which the fluorescence signal by FRET changes in the desired range of ATP concentration can be prepared.

  In the present specification, the “fluorescence signal” includes, but is not limited to, fluorescence intensity, fluorescence intensity ratio, fluorescence lifetime. The fluorescent signal can be measured by a known method. Fluorescent signals can be measured using commercially available devices such as, but not limited to, spectrophotometers, fluorescence lifetime measuring devices and the like.

  In one example, the fluorescent signal is a fluorescent signal from the heart of a non-human transgenic animal expressing the fusion protein. In this example, the fluorescent signal includes, but is not limited to, the fluorescent signal from the entire exposed cardiac surface of the non-human transgenic animal thoracotomy, or a portion of the heart (e.g., atrial portion or The fluorescence signal from the ventricle). The fluorescence signal from a part of the heart is, for example, a fluorescence signal from a region portion in which the fluorescence signal from the entire surface of the heart is measured for a predetermined time and then the change in the fluorescence signal is large within the measurement time. Extraction of such a region portion where the fluorescence signal change is large may be performed automatically or semi-automatically using a program, or may be performed by an analyst.

In another example, the fluorescent signal is a fluorescent signal from cardiomyocytes expressing the fusion protein.
In the present specification, the “cardiomyocyte” expressing the fusion protein includes, but is not limited to, a non-human transgenic animal-derived cardiomyocyte expressing the fusion protein, and a stem cell-derived myocardium expressing the fusion protein. Cells. In this example, the stem cell-derived cardiomyocytes include, but are not limited to, cardiomyocytes made from embryonic stem cells expressing the fusion protein, or made from induced pluripotent stem cells expressing the fusion protein. Cardiomyocytes. In one example, the induced pluripotent stem cells expressing the fusion protein are induced pluripotent stem cells (human iPS cells) derived from human somatic cells according to a known method.

  The conditions for measuring the fluorescence signal are not limited, but are appropriately set by those skilled in the art so as not to interfere with the physiological function of the heart or cardiomyocytes. In one example, the fluorescence signal is obtained by irradiating excitation light for 1 second and photographing by exposure at 10-second intervals for 60 minutes.

  In one example, the fluorescence signal derived from the fusion protein is obtained from a fluorescent protein as a donor and a fluorescent protein as an acceptor by irradiating a fluorescent protein as a donor with excitation light when FRET occurs. be able to. In one example, the fluorescent signals derived from two types of fluorescent proteins can be measured by spectrally splitting the fluorescent signals derived from each fluorescent protein according to a known method. The FRET efficiency can be calculated from the measured fluorescence signal derived from each fluorescent protein.

  In the present specification, “FRET efficiency” means a ratio of energy absorbed by a fluorescent protein serving as a donor to transfer to a fluorescent protein serving as an acceptor. In one example, the FRET efficiency is the ratio of the fluorescence intensity derived from the fluorescent protein as the acceptor (fluorescence intensity on the acceptor side) to the fluorescence intensity derived from the fluorescent protein as the donor (“fluorescence intensity on the donor side”) (= [ The fluorescence intensity on the acceptor side] / [fluorescence intensity on the donor side]).

  In another example, FRET efficiency is the fluorescence lifetime of a fluorescent protein as a donor. In other examples, the acceptor side fluorescence intensity or the donor side fluorescence intensity may vary to reflect FRET efficiency. Therefore, the fluorescence intensity on the acceptor side or the fluorescence intensity on the donor side can be used as the apparent FRET efficiency.

  As used herein, "non-human transgenic animal" means an animal excluding humans, which is produced so as to express the fusion protein. The non-human transgenic animal can be obtained by, but not limited to, integrating the DNA encoding the fusion protein into the chromosome of the non-human animal and expressing the fusion protein.

  The DNA encoding the fusion protein is expressed under the control of a promoter so that it functions in, but is not limited to, cells of non-human transgenic animals. Examples of the promoter include, but are not limited to, β-actin promoter, CMV promoter, and CAG (CAGGS) promoter. In one example, the promoter is, for example, the CAG (CAGGS) promoter from the viewpoint of expression efficiency. In another example, the promoter is a time-specific promoter or a tissue-specific promoter.

  When the DNA encoding the fusion protein is replaced by an endogenous gene on the chromosome by homologous recombination, the DNA includes, but is not limited to, sequences homologous to the site of interest on the chromosome. In one example, the site of interest on the chromosome is the Rosa26 locus. The Rosa26 locus includes, but is not limited to, the human Rosa26 locus (Irion S, et al., Nature Biotechnology volume 25, pages 1477-1482 (2007)), the mouse Rosa26 locus, and corresponding genes in other animals. There is a seat. The mouse ROSA26 locus was discovered by Friedrich and Soriano in 1991 by gene trap experiments with retrovirus-infected embryonic stem (ES) cells (Friedrich, G. and P. Soriano, Genes & Development, 1991,). 5, 1513-1523).

  The DNA encoding the fusion protein may include a recognition sequence represented by a lox-type sequence recognized by a recombinant protein such as, but not limited to, Cre recombinase or Flp recombinase. In one example, a stop codon and / or a drug resistance gene is arranged between the promoter and the DNA encoding the fusion protein, and when Cre recombinase is expressed, the promoter and the DNA encoding the fusion protein are directly linked, Transcription can be initiated. Examples of the lox-based array include loxP, lox71, lox66, lox511, lox2272, Vlox (VCre), and Slox (SCre). In other examples, FRT sequences recognized by FLP may be used as recognition sequences.

  In one example, the DNA encoding the fusion protein contains a selectable marker. "Selectable marker" means a genetic element that provides a selectable phenotype to cells into which the selectable marker has been introduced. Selectable markers are, but are not limited to, genes encoding proteins that confer resistance to agents that inhibit cell growth or kill cells. Any known selection marker can be used without particular limitation. Examples of the selection marker include Neo gene, Hyg gene, hisD gene, Gpt gene, and Ble gene. Drugs useful for selecting the presence of a selectable marker include, but are not limited to, G418 for the Neo gene, hygromycin for the Hyg gene, histidinol for the hisD gene, and Gpt gene. For 6-thioxanthine, and for the Ble gene bleomycin.

Examples of the non-human transgenic animal include WO 2015/108102, Hogan et al., Manipulating the Mouse Embryo: A Laboratory Manual, Cold Spring Harbor Laboratory (1986), US Pat. It can be prepared according to the method described in No. 750,826. Specifically, the non-human transgenic animal can be obtained by a method including the following steps (a) to (g):
(A) introducing a targeting vector containing a DNA encoding the fusion protein into an embryonic stem (ES) cell of a non-human animal; (b) an ES cell having a chromosome into which the DNA encoding the fusion protein is inserted. (C) introducing the selected ES cells into a fertilized egg; (d) transplanting the fertilized egg into the oviduct or uterus of a pseudopregnant female non-human animal; (e) the non-human animal A step of selecting a germline chimera from the bred and born pups; (f) mating the germline chimera, and selecting an individual having a chromosome into which the DNA encoding the fusion protein is inserted from the pups born Step: (g) The individuals obtained in step (f) are crossed with each other, and the DNA encoding the fusion protein is inserted into both homologous chromosomes from the offspring. Selecting a homozygote was.

  In one example, the non-human transgenic animal has a DNA encoding the fusion protein inserted into the ROSA26 locus on its chromosome by a genetic engineering technique. In another example, the non-human transgenic animal is genetically engineered to have the DNA encoding the fusion protein operably linked to a CAG promoter sequence. In another example, the non-human transgenic animal has a DNA encoding the fusion protein inserted into the ROSA26 locus on its chromosome by a genetic engineering technique, and the DNA is operably linked to a CAG promoter sequence. Has been done.

  The non-human transgenic animal is, for example, a mouse, rat, rabbit, goat, pig, dog, cat, guinea pig, hamster, sheep, cow, marmoset transgenic animal. In one example, the non-human transgenic animal is a transgenic mouse, transgenic rat or transgenic rabbit. In another example, the non-human transgenic animal is a transgenic mouse.

  The non-human transgenic animal may be a disease model non-human transgenic animal produced according to a known method. Disease model non-human transgenic animals include, but are not limited to, diabetes model animals, cancer disease model animals, and infectious disease model animals.

  In the present specification, the “stem cell” expressing the fusion protein includes, but is not limited to, embryonic stem cells and induced pluripotent stem cells produced so as to express the fusion protein. Although not limited, the stem cell has a DNA encoding the fusion protein inserted into its chromosome by a genetic engineering technique. In one example, the stem cell is inserted at the ROSA26 locus on its chromosome and the DNA is operably linked to a CAG promoter sequence. The stem cells can be produced, for example, according to the method described in International Publication WO2015 / 108102. The induced pluripotent stem cells can be prepared by, but not limited to, introducing a reprogramming factor into somatic cells expressing the fusion protein.

  Examples of the stem cells include human, mouse, rat, rabbit, goat, pig, dog, cat, guinea pig, hamster, sheep, cow, and marmoset-derived cells. In one example, the stem cells are human-derived cells. In one example, the stem cells are induced pluripotent stem cells derived from human somatic cells. The method for producing cardiomyocytes from stem cells is not limited, but a method for inducing differentiation of cardiomyocytes using a cytokine in the embryoid body formation method (for example, Nature. 2008 May 22; 453 (7194): 524-8). ), A method of inducing differentiation of cardiomyocytes without using cytokines in adherent culture (for example, Proc Natl Acad Sci USA., 2012 July 3; 109 (27): E1848-57), combined use of adherent culture and suspension culture, A method for inducing differentiation of cardiomyocytes without using cytokines (for example, Cell Rep., 2012 Nov29; 2 (5): 1448-60) is known. In one example, cardiomyocytes are prepared by culturing the stem cells with factors that induce differentiation into cardiomyocytes.

  The “determination” of whether or not the administered test substance causes cardiotoxicity is determined by whether or not the balance between supply and demand (consumption) of ATP in the heart is disturbed. Without being bound by theory, the heart has a pumping action that drives blood into the aorta. The pumping action of the heart is carried by the myocardium. Myocardium needs to supply a large amount of ATP as its energy source, and consumes a large amount of ATP by its pumping action. In the normal heart, ATP supply and demand are considered to be balanced (Fig. 1a), and ATP concentration in the heart is considered to be stable.

  On the other hand, if the supply of ATP is delayed due to, for example, cardiac ischemia, ATP consumption will increase relative to the ATP supply, and the balance between supply and demand of ATP will be disturbed (FIG. 1b). Further, when ATP is less likely to be consumed due to bradycardia or reduced systole, ATP consumption becomes smaller than ATP supply, and the balance between supply and demand of ATP is disturbed (FIG. 1c). When such an imbalance between supply and demand of ATP occurs, it is considered that the ATP concentration in the heart fluctuates. From the above, the determination of whether or not the administered test substance causes cardiotoxicity is determined based on the change in ATP concentration in the heart.

  It is speculated that the balance between supply and demand of ATP is maintained even in beating cardiomyocytes. Therefore, in another example, the determination of whether the administered test substance causes cardiotoxicity depends on whether the balance between supply and demand (consumption) of ATP in cardiomyocytes is disturbed, that is, cardiomyocyte It is determined based on the change in the ATP concentration in.

  In one embodiment, ATP concentration can be calculated from the fluorescent signal. In one example, the ATP concentration is introduced into the cytoplasm of cells expressing the fusion protein to a predetermined concentration, ATP is introduced, and a calibration curve showing the relationship between the fluorescence signal from the cells and the ATP concentration is obtained to obtain the fusion protein. It can be obtained from a fluorescence signal from a cardiomyocyte or heart containing a protein-expressing ATP of unknown concentration and the calibration curve. In this example, the method of introducing a predetermined concentration of ATP into the cytoplasm of the cell is not limited, but by providing a hole in the cell membrane of the cell and adding ATP to the culture solution to a predetermined concentration. Can be implemented.

  In another example, the ATP concentration is determined by measuring a fluorescence signal from the cell expressing the fusion protein, and measuring the ATP concentration in the cell after the measurement, and measuring the fluorescence signal and the ATP concentration in the cell, respectively. Can be obtained from the fluorescence signal from a cardiomyocyte or heart containing ATP of unknown concentration expressing the fusion protein and the calibration curve. In the above example, as a method for measuring the ATP concentration of cells, a known method, for example, the luciferase method can be used.

  In order to obtain the ATP concentration from the calibration curve obtained as described above, a function obtained by fitting a model formula to the calibration curve may be used instead of using the calibration curve described above. The model formula is, for example, Hill's formula. In this example, the ATP concentration of the sample can be calculated by applying a fluorescence signal obtained from a cardiomyocyte or heart of unknown concentration to the function obtained by fitting.

In one embodiment, the determination of whether the administered test substance causes cardiotoxicity is made by comparing the measured fluorescence signal with a first threshold. In this embodiment, the fusion protein results in FRET upon binding of ATP.
As an example, the measured fluorescence signal is the FRET efficiency, and the first threshold value is a numerical value larger than the maximum value of the FRET efficiency measured when the test substance is not administered (for example, 3 times the maximum value). Is. If the measured FRET efficiency is greater than or equal to the first threshold, the administered test substance is determined to cause cardiotoxicity. In this example, the administered test substance is determined not to be cardiotoxic if the measured fluorescence signal is below the first threshold.

  In another example of the above embodiments, determining whether the administered test substance causes cardiotoxicity further comprises comparing the measured fluorescence signal with a second threshold that is less than the first threshold. .. As an example, the second threshold value is a numerical value smaller than the minimum value of the FRET efficiency measured when the test substance is not administered. If the measured FRET efficiency is below the first threshold value and above the second threshold value, it is determined that the administered test substance does not cause cardiotoxicity. In this example, the administered test substance is determined to be cardiotoxic if the measured fluorescence signal is greater than or equal to the first threshold and less than the second threshold.

  In the above embodiment, the fluorescence signal measured was the FRET efficiency, but the present aspect is not limited thereto. For example, the measured fluorescence signal may be an integrated value of the FRET efficiency in the predetermined time section [a, b] of the function (f (x)) representing the FRET efficiency with respect to the measurement time.

  In the above embodiment, both the first threshold value and the second threshold value are FRET efficiency, but the present aspect is not limited to this. For example, the first threshold may relate to FRET efficiency and the second threshold may be an integral value of FRET efficiency. Although the above embodiment includes comparison with one threshold value or two threshold values, the present aspect is not limited thereto. In other embodiments, three thresholds may be compared. In the above-described embodiment, the fusion protein is one in which FRET is generated by binding ATP, but the present aspect is not limited thereto. For example, the fusion protein may be one in which FRET is eliminated by binding ATP.

  In one aspect, a method of determining whether a test substance produces cardiotoxicity is provided. The determination method is a step of administering a test substance to a non-human transgenic animal expressing a fusion protein containing an ATP binding moiety, a fluorescent protein as a donor for fluorescence resonance energy transfer, and a fluorescent protein as an acceptor. Measuring the fluorescence signal from the heart of the non-human transgenic animal; and determining whether the test substance causes cardiotoxicity based on the measured fluorescence signal. As described in the present specification, the characteristics regarding the test substance, the characteristics regarding the fusion protein, the characteristics regarding the step of administering to a non-human transgenic animal, and the characteristics regarding the step of measuring the fluorescence signal from the heart of the non-human transgenic animal, etc., This applies to this aspect.

  In the above aspect, the non-human transgenic animal expressing the fusion protein can be replaced with a non-human chimeric animal having a heart containing cardiomyocytes expressing the fusion protein. In this embodiment, the step of administering a test substance to a non-human chimeric animal expressing the fusion protein; the step of measuring a fluorescent signal from the heart of the non-human chimeric animal; and the test substance based on the measured fluorescent signal. A determination method is provided that includes a step of determining whether or not causes cardiotoxicity. The characteristics of the step of administering a test substance to a non-human transgenic animal described in the present specification, the characteristics of measuring a fluorescence signal from the heart of the non-human transgenic animal, and the like are non-human transgenic animals. The present invention is also applied to this embodiment after being read as a human chimeric animal.

  As used herein, the term “non-human chimeric animal” refers to an animal in which cells expressing the fusion protein and non-human cells heterologous to the cells coexist, and includes cardiomyocytes derived from cells expressing the fusion protein. It means an animal with a heart. The non-human chimeric animal can be produced by a known developmental engineering method (for example, Wu et al., 2017, Cell, 168, 473-486). In one example, a non-human chimeric animal is prepared by injecting a stem cell expressing the fusion protein into an embryo composed of a non-human stem cell of a species different from the cell, which has been modified by genetic engineering so as not to form a heart. can do. In this example, the stem cells expressing the fusion protein are, for example, embryonic stem cells or induced pluripotent stem cells (eg induced pluripotent stem cells derived from human somatic cells according to known methods (human iPS cells)). You may.

  In one aspect, a method of determining whether a test substance produces cardiotoxicity is provided. In one embodiment, the determination method comprises the step of contacting a test substance with cardiomyocytes; the step of measuring the ATP concentration in the cardiomyocytes; and the test substance causes cardiotoxicity based on the measured ATP concentration. It includes a step of determining whether or not. In another embodiment, the determination method comprises the step of administering a test substance to a non-human animal, and measuring the ATP concentration in the heart of the non-human animal; and the test substance based on the measured ATP concentration. Determining whether or not causes cardiotoxicity. In this aspect, the measurement of ATP concentration in cardiomyocytes or heart can be performed by using a known measurement method (for example, magnetic resonance spectroscopy (MRS)), but is not limited thereto. Characteristics of the test substance, methods of contacting the test substance with cardiomyocytes, features of the cardiomyocytes, and features of determining whether the test substance causes cardiotoxicity based on the ATP concentration, etc. described in the present specification Also applies to this aspect.

  As used herein, “non-human animal” means an animal other than human. Non-human animals are, but are not limited to, vertebrates, especially mammals. Non-human animals are, for example, mice, rats, rabbits, goats, pigs, dogs, cats, guinea pigs, hamsters, sheep, cows or marmosets.

  In the present specification, the “drug candidate substance” means a test substance which is determined not to cause cardiotoxicity by the determination method according to any one of the above aspects. The expression of the judgment result of the step of carrying out the judgment method is not limited to "causes cardiotoxicity" or "does not cause cardiotoxicity". “There is no concern”, “cardiotoxicity is concerned / not concerned”, “drug candidate is / is not”. As used herein, the “drug candidate substance library” includes two or more drug candidate substances (eg, 10 or more, 100 or more, or 1000 or more drug candidate substances).

  In one aspect, a method of producing a drug candidate library is provided. The above-mentioned production method is a step of carrying out the above-mentioned determination method of determining whether or not cardiotoxicity is caused for each of two or more test substances used in the production of a library of drug candidate substances; The step of selecting a test substance determined not to cause cardiotoxicity and producing a drug candidate substance library is included. The features described above regarding the determination method and the like in the present specification are also applied to the determination method and the like of this aspect.

  In one embodiment, a fusion protein comprising an ATP binding moiety, a fluorescent protein as a donor for fluorescence resonance energy transfer, and a fluorescent protein as an acceptor for determining whether a test substance causes cardiotoxicity. Expressing cardiomyocytes, non-human chimeric animals or non-human transgenic animals are provided. The features described above regarding the determination method and the non-human transgenic animal are also applied to the cardiomyocyte, the non-human chimeric animal, and the non-human transgenic animal of the present embodiment.

  Hereinafter, specific examples will be described, but they show preferred embodiments of the present invention, and do not limit the invention described in the appended claims in any way.

[Comparative Example 1]
(1) Transgenic Mouse Expressing Fusion Protein A transgenic mouse that was created according to the method described in International Publication WO2015 / 108102 and emits different color fluorescence depending on the ATP concentration was used.

  The transgenic mouse comprises a Bacillus subtilis-derived ATP synthase ε subunit (R122K / R126K), a green fluorescent protein (cp173-mEGFP) at its N-terminus, and a red fluorescent protein (mKusabilaOrange) at the C-terminus of the ε subunit. ) Is fused with (GO-ATeam2 (Nakano, M, ACS Chem Biol, 2011; 6,709-715).) The DNA encoding the fusion protein is expressed in the transgenic mouse. It is inserted at the ROSA26 locus on the chromosome and is operably linked to the CAG promoter sequence.

  In the transgenic mouse, ATP binding to the epsilon subunit portion in the fusion protein results in FRET between the two fluorescent proteins fused in the fusion protein. This FRET causes the fusion protein to emit fluorescent signals of different colors before and after ATP binding. Specifically, the fusion protein mainly emits a green (peak wavelength: 509 nm) fluorescent signal before ATP binding, and mainly emits a red (peak wavelength: 559 nm) fluorescent signal after ATP binding. ATP and the ε subunit are bound in an ATP concentration-dependent manner. By measuring the fluorescence signal or the change in the fluorescence signal emitted from the whole heart of the transgenic mouse or a part thereof, the change in the ATP concentration or the ATP concentration of the heart of the mouse or a part thereof can be examined.

(2) Fluorescence image measurement The transgenic mouse was anesthetized with isoflurane and then tracheal intubated. Physiological saline (0.9% NaCl aqueous solution) was administered through the jugular vein at a flow rate of 15 μL / min. The anesthetized transgenic mouse was subjected to a thoracotomy to expose the heart. The exposed heart is irradiated with excitation light from the light source Xcite LED 110 through the excitation filter 470/40 nm, and the fluorescence signal from the heart is converted to Dual-View (Nippon Roper Co., BP515 / 30, DM540, BP575 / 40). Then, the fusion protein was separated into a fluorescent signal (G) derived from a green fluorescent protein and a fluorescent signal (R) derived from a red fluorescent protein. Each fluorescence image of the heart was taken with ORCA-Flash 4.0 (Hamamatsu Photonics).

  The fluorescence images obtained at the start of imaging (0 min) were overlaid (Fig. 2a). Excitation light was applied only during imaging. Photographing was performed for 60 minutes at 10 second intervals with an exposure time of 1 second (FIG. 2b).

(3) Electrocardiogram and rectal temperature measurement Electrocardiogram and rectal temperature were measured in parallel with imaging of the heart. The electrocardiogram showed no significant change between before and after administration of physiological saline (FIG. 2c). There was also no significant change in rectal temperature.

(4) ATP concentration calculation (4.1) Fluorescence intensity ratio and ATP concentration in fibroblasts Fibroblasts were established from the transgenic mouse expressing the fusion protein. Α-toxin was added to the culture solution of the fibroblasts in order to provide pores in the cell membrane of the established fibroblasts. Then, ATP was added to the culture solution so as to have various concentrations and cultured for about 10 minutes, and then a fluorescence image of the fibroblast was obtained. The fluorescence intensity ratio of the fluorescence intensity (R) derived from the red fluorescent protein to the fluorescence intensity (G) derived from the green fluorescent protein (= [fluorescence intensity (R)] / [fluorescence intensity (G)]) at each ATP concentration Calculated. A scatter diagram was prepared by plotting the calculated fluorescence intensity ratio against the ATP concentration.

(4.2) Fluorescence intensity ratio and ATP concentration of fertilized eggs Fertilized eggs were collected from transgenic mice expressing the fusion protein. A culture solution containing the collected fertilized eggs was placed on a slide glass, and a glycolytic inhibitor (2-deoxy-D-glucose; 2DG) and an electron transfer inhibitor (Antimycin A) were added to the culture solution. .. After the addition of the inhibitor, an image was taken when the ratio of fluorescence intensity obtained from the fertilized egg changed, and immediately after that, the fertilized egg was physically disrupted. The volume of fertilized eggs was estimated before crushing. The ATP concentration in the disrupted liquid of fertilized eggs was quantified by the Luciferase method. The quantified ATP concentration of the disrupted liquid and the ATP content of the fertilized egg were calculated from the disrupted liquid, and the ATP concentration of the fertilized egg was calculated from the calculated ATP content and the volume of the fertilized egg. A scatter plot was prepared by plotting the fluorescence intensity ratio of each fertilized egg against the ATP concentration of the fertilized egg.

(4.3) Fluorescence intensity ratio and ATP concentration Shown in the scatter plot created in (4.2) above and the curve of fluorescence intensity ratio and ATP concentration shown in the scatter plot created in (4.1) above. The obtained fluorescence intensity ratio and the curve of the ATP concentration almost matched. Therefore, from the fluorescence intensity ratio and ATP concentration data obtained in (4.1) and the fluorescence intensity ratio and ATP concentration data obtained in (4.2), a scatter diagram of each ATP concentration and fluorescence intensity ratio It was created.

Against the curve of the fluorescence intensity ratio and ATP concentrations indicated in scatter diagram created, Hill equation _{[R = (R max -R min} ) × [ATP] n / [ATP] n + K d n) + R min [Wherein R is the fluorescence intensity ratio, R _max is the maximum fluorescence intensity ratio, R _min is the minimum fluorescence intensity ratio, and Kd is the ε subunit of the fusion protein and ATP] Dissociation constant (1.6 [nM]), and [ATP] is the ATP concentration. ]. By fitting,
was gotten.

  The colors of the superimposed fluorescence images reflect the fluorescence intensity ratio of the fluorescence intensity (R) derived from the red fluorescent protein to the fluorescence intensity (G) derived from the green fluorescent protein. By using (Equation 1), the ATP concentration is calculated from the fluorescence intensity ratio of the region of interest (ROI) of each image. A bar showing the relationship between the color change and the ATP density is shown on the right side of the superposed images of FIGS. 2a and 2b.

(5) ATP Concentration Change ATP concentration change (ΔATP (mM) = [ATP concentration at measurement (min) (mM) from the start of measurement (0 minutes) immediately after administration of physiological saline to continuous administration for 60 minutes) )]-[ATP concentration (mM) at the start of measurement (0 minutes)]) (Fig. 2e). FIG. 2e shows that in the measurement system of Comparative Example 1, the ATP concentration is stable within a fluctuation range of about 0.05 mM.

[Example 1]
The electrocardiogram measurement (FIG. 3a), the fluorescence image acquisition (FIG. 3b), and the ATP concentration were measured in substantially the same manner as in Comparative Example 1 except that disopyramide was administered through the jugular vein. Figure 3c).

  Figure 3a shows that 0.2 [mg / ml] disopyramide shows no significant changes in electrocardiogram. Figures 3b and 3c show that administration of disopyramide increased the ATP concentration in the heart over time.

  Disopyramide is an antiarrhythmic drug (Class 1a arrhythmic agent) used to treat ventricular tachycardia by blocking Na channels and reducing myocardial contractility. It is known that disopyramide presents with repolarization abnormality (QT prolongation) accompanied by T-wave morphological abnormality in the electrocardiogram, and may cause severe ventricular arrhythmia such as torsade de pointe (TdP), resulting in sudden death. There is.

  In Example 1, even with disopyramide at a concentration at which no QT prolongation was observed from the electrocardiogram (Fig. 3a), a significant change in ATP concentration in the heart was detected (Figs. 3b and c). This result indicates that, according to the invention of the present embodiment, it is possible to find a substance capable of causing QT prolongation syndrome even under the administration conditions of a test substance in which QT prolongation is not observed in the electrocardiogram. Indicates.

[Examples 2 to 14]
The time-dependent change in ATP concentration in the heart was measured in substantially the same manner as in Example 1 except that the test substances in the table below were administered.

  In Examples 2 to 14, in the measurement of the ATP concentration change for 60 minutes, the value of the ATP concentration change three times the maximum value (about 0.05 mM, Comparative Example 1) of the ATP concentration change when physiological saline was used. (0.15 mM) as the first threshold value, the maximum value of ATP concentration change was less than the first threshold value, the test substance was judged not to cause cardiotoxicity, and the test value was above the first threshold value. The substance is determined to cause cardiotoxicity. In the present embodiment, a substance determined not to cause cardiotoxicity is specified as a drug candidate substance.

In all of the test substances of Examples 2 to 11, the maximum value of the change in ATP concentration was the first threshold value (0.15 mM) or more (FIGS. 4 and 5), and it was determined that cardiotoxicity might occur. ..
It was determined that all the test substances of Examples 12 to 14 had the maximum change in ATP concentration less than the first threshold value (FIG. 6) and did not cause cardiotoxicity.

  The maximum value of ATP concentration change of the test substance of Example 1 was not less than the first threshold value (0.15 mM) (FIG. 3), and it was determined that cardiotoxicity might occur. Therefore, in the present embodiment, it was determined that the test substances of Examples 1 to 11 among the test substances of Examples 1 to 14 may cause cardiotoxicity.

  The candidate substances of Examples 1 to 11 are registered with the US Food and Drug Administration (FDA) as compounds capable of producing TdP. It was determined that the candidate substances of Examples 1 to 11 may cause cardiotoxicity also in this embodiment. These results indicate that, according to the present embodiment, a drug candidate substance library can be produced by removing a test substance determined to cause cardiotoxicity from the test substance library. ..

In the present embodiment, it was determined that the test substances of Examples 12 to 14 among the test substances of Examples 1 to 14 did not cause cardiotoxicity.
It is known that the test substances of Examples 12 and 13 experimentally have no effect on repolarization of the heart or that they do not prolong the QT interval. Also in this embodiment, it was determined that the compounds of Examples 12 and 13 did not cause cardiotoxicity. These results indicate that according to this embodiment, a drug candidate substance library can be produced by collecting substances that do not cause cardiotoxicity.

[Examples 15 to 17]
The time-dependent change in ATP concentration in the heart was measured in substantially the same manner as in Example 1 except that the test substances in the table below were administered.

  In Examples 15 to 17, 0 (zero) was set as the second threshold value for the integral value of the function (f (x)) indicating the change in the ATP concentration in the interval [0, 60]. In the present embodiment, the test substance that is less than the second threshold value is determined to be likely to cause cardiotoxicity.

  As can be seen from FIGS. 7A to 7C, the integrated value in the section [0, 60] of the function showing the change in the ATP concentration in Examples 15 to 17 was a negative (<0) value. Therefore, in the present embodiment, it was determined that all the test substances of Examples 15 to 17 may cause cardiotoxicity.

  It is known that all the test substances of Examples 15 to 17 can exhibit abnormal electrocardiogram and ventricular dysfunction. Also in this embodiment, it was determined that all of Examples 15 to 17 might cause cardiotoxicity. These results indicate that, according to this embodiment, it is useful for identifying a substance that may cause cardiotoxicity from a test substance library.

[Example 18]
In Examples 1 to 17, changes in ATP concentration in the whole heart of the transgenic mouse were measured. In Example 18, changes in the ATP concentration in the ventricle part of the test substance ifosfamide were measured (FIG. 7c, left ventricle part). For the test substance ifosfamide, the change in the ATP concentration in the ventricle (Fig. 7c, left ventricle) was larger than that in the case where the change in the ATP concentration in the whole heart was measured (Fig. 7c, whole heart). I was able to measure. This result indicates that by measuring the change in ATP concentration in a specific part of the heart, it is possible to more sensitively determine whether or not the test substance may cause cardiotoxicity.

Claims (9)

A method for determining whether a test substance causes cardiotoxicity,
Administering a test substance to a non-human transgenic animal expressing a fusion protein comprising an ATP binding moiety, a fluorescent protein as a donor for fluorescence resonance energy transfer, and a fluorescent protein as an acceptor;
A method for determining, comprising: measuring a fluorescent signal from the heart of the non-human transgenic animal; and determining whether the test substance causes cardiotoxicity based on the measured fluorescent signal. A method for determining whether a test substance causes cardiotoxicity,
Contacting the test substance with cardiomyocytes expressing a fusion protein comprising a fusion protein comprising an ATP binding moiety, a fluorescent protein as a donor for fluorescence resonance energy transfer, and a fluorescent protein as an acceptor;
A method for determining, comprising a step of measuring a fluorescent signal from the cardiomyocyte; and a step of determining whether or not the test substance causes cardiotoxicity based on the measured fluorescent signal.   The ATP-binding portion is a CBS domain of inosine monophosphate dehydrogenase 2 (IMPDH2) or a fragment thereof or a modified form thereof, GlnK1 or a fragment or a modified form thereof, or an ε subunit of ATP synthase or a fragment or a modified form thereof. The determination method according to claim 1, which is a body.   The determination method according to claim 1, wherein the ATP-binding portion is an ε subunit of ATP synthase, a fragment thereof, or a modified form thereof.   In the fusion protein, one of the fluorescent protein as the donor and the fluorescent protein as the acceptor is linked to the amino-terminal side of the ATP-binding portion, and the other is linked to the carboxy-terminal side of the ATP-binding portion. The determination method according to claim 1, which is performed.   The cardiomyocyte is a cardiomyocyte derived from a non-human transgenic animal that expresses the fusion protein, or a cardiomyocyte prepared from a stem cell that expresses the fusion protein. Judgment method. A method for determining whether a test substance causes cardiotoxicity,
A step of contacting a test substance with cardiomyocytes, and a step of measuring the ATP concentration in the cardiomyocytes; or a step of administering the test substance to a non-human animal, and a step of measuring the ATP concentration in the heart of the non-human animal ,
The determination method further comprising the step of determining whether or not the test substance causes cardiotoxicity based on the measured ATP concentration. A method for producing a drug candidate substance library, comprising:
A step of carrying out the determination method according to any one of claims 1 to 7 for determining whether or not each of two or more test substances causes cardiotoxicity; and, in the step, no cardiotoxicity occurs. A method for producing, comprising a step of selecting a test substance determined to be and producing a drug candidate substance library.   Cardiomyocytes expressing a fusion protein containing an ATP binding moiety, a fluorescent protein as a donor for fluorescence resonance energy transfer, and a fluorescent protein as an acceptor for determining whether a test substance causes cardiotoxicity, or Non-human transgenic animal.