JP2008118882A - Danio rerio showing torsades de pointes type ventricular tachycardia - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for preparing a model animal of torsades de pointes type ventricular tachycardia by a simple and inexpensive method. <P>SOLUTION: This method for producing Danio rerio showing the torsades de pointes type ventricular tachycardia is characterized by rearing the Danio rerio fertilized eggs within 6 days after the fertilization under the presence of a QT-elongating agent and rearing in the absence of the QT-elongating agent after developing ventricular interruption. Also, a method for evaluating the possibility of a test substance inducing the torsades de pointes type ventricular tachycardia, is disclosed. The Danio rerio showing the torsades de pointes type ventricular tachycardia is useful as the model animal for the research of morbidity of arrhythmia and for the research and development of a treating and preventing agent of the arrhythmia. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a zebrafish useful as a model animal exhibiting a torsade de pointes ventricular tachycardia.

  One of the most serious side effects caused by pharmaceuticals is drug-induced arrhythmia (QT prolongation syndrome, AV block, etc.). For example, the antihistamine terfenadine, withdrawn from the market in 1998, developed a high rate of drug-induced QT syndrome in patients who took it, some of which developed into Torsades de Pointes, suddenly To death. In addition to terfenadine, drugs that withdrew from the pharmaceutical market due to this side effect include the antihistamine agent astemizole and the gastrointestinal motility promoter cisapride. Similar side effects have also been reported with some other psychotropic drugs and antibiotics.

QT prolongation syndrome is a pathological condition in which the QT interval (the time required from ventricular depolarization to repolarization) becomes longer on the electrocardiogram, and the corrected QT time (QTc = QT / (RR) ^1/2 , RR is an RR interval) is 440 msec for men and 460 msec or more for women. Among arrhythmias that exhibit QT prolongation, it is known that Torsade de Pointes ventricular tachycardia (hereinafter also referred to as Torsade de Pointes) has a high risk of sudden death. Torsade de pointes is a polymorphic ventricular tachycardia with QT prolongation and is characterized by an electrocardiogram waveform that varies so that the amplitude of the QRS wave twists around the baseline.

  Drug-induced long QT syndrome, especially Torsade de Pointes, is a lethal side effect and the impact on drug therapy is enormous. As model animals exhibiting symptoms of QT prolongation syndrome, monkeys, dogs, rats, mice and the like are used. However, mammalian model animals are expensive and have a drawback of requiring a large experimental facility. In recent years, zebrafish has been attracting attention as a model animal that is cheaper and suitable for a large amount of treatment. The greatest advantage of zebrafish is that they are vertebrates that have a fast ontogeny and can be used for large-scale drug screening. Therefore, if a zebrafish model animal exhibiting symptoms of QT prolongation syndrome can be developed, it is considered to be very useful as a system for studying the pathophysiology of arrhythmia and the side effects of drugs.

Langheinrich and colleagues found that exposure of early zebrafish embryos to various QT prolonging agents resulted in an AV-blocking bradyarrhythmia in the zebrafish heart and identified the gene Herg (Zerg) involved in this event. (Langheinrich, et al., Toxicol Appl Pharmacol. 2003 Dec 15; 193 (3): 370-82.). In this paper, exposure of zebrafish embryos to QT prolongers did not cause tachyarrhythmia or torsade de pointes arrhythmia because of differences in physiology and channel structure between embryos and adults Or maybe due to species differences.
Langheinrich, et al., Toxicol Appl Pharmacol. 2003 Dec 15; 193 (3): 370-82.

  An object of the present invention is to provide a method for producing a model animal of torsade de pointes ventricular tachycardia by a simple and inexpensive method.

  The present inventors administered zebrafish to 92 types of drugs that have been reported to have proarrhythmic effects in humans, and analyzed the effects on cardiac function and electrocardiogram, and found that waveform abnormalities very similar to those in humans. We found that it was also recognized in zebrafish. Specifically, exposure of fertilized eggs of zebrafish to QT prolongation causes cardiac arrest, and subsequent washing of QT prolongation restores heart rate, and during this recovery, Torsade de Pointes ventricular frequency It was found that the ECG waveform characteristic of the beat was shown.

  The present invention is a method for producing a zebrafish exhibiting a torsade de pointes type ventricular tachycardia, wherein a zebrafish fertilized egg within 6 days after fertilization was bred in the presence of a QT prolongation drug, resulting in ventricular arrest. There is provided a method characterized in that the animal is later raised in the absence of a QT prolonging agent.

  The present invention is also a method for evaluating the possibility that a test substance induces torsades de pointes ventricular tachycardia in humans, wherein zebrafish fertilized eggs within 6 days after fertilization are bred in the presence of the test substance. If the animal is bred in the absence of the test substance after ventricular arrest has occurred, and then the zebrafish electrocardiogram is measured after ventricular pulsation is restored, is the torsade de pointes ventricular tachycardia shown? A method is provided that determines whether or not.

  Zebrafish (genus name Danio, for example Danio rerio) is a small tropical fish of about 5 cm in length native to India. It belongs to the family Carpidae, Carpaceae, Danio subfamily (both Lasbola subfamily and Haejako subfamily). And close to goldfish. Because of the scarlet horizontal stripes on the adult body surface, it has the name of a zebra. It is a fish that is easy to breed and breed, has a low distribution price, and is often kept as an ornamental fish. Several inbred lines are recognized internationally, but as a species used in the present invention, a golden line with few horizontal stripes is desirable. Zebrafish is a fish, but the development and structure of major organs and tissues are very similar to those of humans, and the process in which each part (organ, etc.) differentiates and forms from a fertilized egg can be observed through a transparent body. . Even if the basic structures of major organs and tissues are completed 48 hours after fertilization, the body length is 2 mm or less and it can be handled in a small space such as a 96-well plate. Therefore, it can be applied to screening of new drug candidate compounds using the influence on the whole model animal as an index, and screening of new drug candidate compounds using zebrafish has already been carried out commercially. The entire zebrafish genome has also been decoded (Sanger Institute), and gene expression profiles can be analyzed.

  As water for breeding fertilized eggs of zebrafish, any breeding water suitable for breeding tropical fish may be used. For example, a breeding solution having the following composition (calcium chloride 0.1 g, sodium chloride 3.5 g , Potassium chloride 0.05 g, sodium hydrogen carbonate 0.2 g / 1 L), Instant Ocean (final concentration 60 μg / ml), and the like can be used. The breeding temperature is generally 26 ° C. to 30 ° C., preferably about 28 ° C. As the container, for example, a wide range of experimental containers from 96 well microplates to 100 mL beakers can be used. The amount of water per animal is at least 100 μl in the fertilized egg state.

  When fertilized eggs are bred in this manner, they usually hatch in about 48 to 72 hours immediately after fertilization, and the development process is almost completed. The body length at this time is about 3 mm. Furthermore, if the breeding is continued under similar conditions, formation of most organs other than bone is completed in about 5 days, and formation of all organs including bone is completed in about 6 days. The body length becomes about 5 mm in about 7 days, and the size of the heart is about 0.3 mm at this time. Since zebrafish is transparent until about one month after fertilization, the morphology of various organs including the heart and blood vessels can be observed with the naked eye.

  In the method of the present invention, first, zebrafish fertilized eggs within 6 days after fertilization are bred in the presence of a QT prolonging agent. If it exceeds 6 days after fertilization, an individual who does not receive the action of the QT prolonging agent will be produced, and the test results will vary. Preferably, a fertilized egg 48 to 72 hours after fertilization is used. Particularly preferably, the QT prolonging agent is exposed 72 hours after fertilization. It is also possible to expose to a QT prolonging agent immediately after fertilization or 24 hours after fertilization. When a fertilized egg is bred in the presence of a QT prolonging agent, abnormal pulsations are observed after 6 to 24 hours, and it is observed that AV block is generated. Furthermore, if the breeding is continued in the presence of a QT prolonging drug, ventricular arrest is reached after 24 to 48 hours. Next, if the breeding water is changed within 24 hours after the ventricular arrest and the breeding is continued in the absence of the QT prolonging drug, the heart rate is recovered within 6 to 24 hours. During this recovery, individuals showing an electrocardiogram waveform characteristic of torsade de pointes ventricular tachycardia can be seen.

  As the QT prolongation drug, any drug that is known to cause the QT prolongation syndrome may be used. For example, terfenadine, astemizole, cisapride, quinidine, procainamide, disopyramide, cibenzoline, Examples include propafenone, amiodarone, sotalol, pepridyl, amitriptyline, imipramine, nortriptyline, maprotiline, chlorpromazine, haloperidol, erythromycin, clarithromycin, doxorubicin and the like. Among the drugs currently on the US market, those having a QT prolonging action are described in the following URL, and these can also be used as QT prolonging drugs in the present invention: http: //www.arizonacert. org / medical-pros / drug-lists / drug-lists.htm #).

  Preferably, astemizole is used as the QT extender. Astemizole is a trade name of hismanal, structural formula 1-[(4-fluorophenyl) methyl] -N- [1- [2- (4-methoxyphenyl) ethyl] -4-piperidyl] -benzimidazole- 2-amine, C28H31FN4O, a compound with a molecular weight of 458.571. Astemizole is a long-acting histamine (H1) receptor antagonist with no sedation, but is not currently used as a pharmaceutical because of side effects such as causing arrhythmia.

  The preferred concentration of astemizole in the breeding water is 100 nM to 10 μM, more preferably 300 nM to 1 μM. If the concentration is too low, the appearance frequency of arrhythmia is low, and if the concentration is too high, the frequency of non-specific changes and death increases, which is inappropriate for the preparation of model animals.

  In addition, astemizole is microinjected into fertilized eggs, microinjection into each tissue by injection into fry, oral administration to fry (after 4 days after fertilization), administration to parent fish (oral or dissolution, to each tissue) The zebrafish or a fertilized egg thereof may be administered by injection).

  The model animal of torsade de pointes ventricular tachycardia obtained by the method of the present invention is useful as a system for studying the pathophysiology of arrhythmia and the side effects of drugs. Because zebrafish is transparent, the zebrafish heart morphology can be easily observed with the naked eye or with a stereomicroscope. In order to measure the heart rate of the atrium and the ventricle, the number of processes from contraction to relaxation in each of the atrium and the ventricle in 15 seconds is measured using a counter under a microscope. In addition, in order to measure an electrocardiogram, a reference electrode is placed in a conductive solution in the measurement unit, and the electrical difference from the electrode in contact with the living body is recorded. Signal. Furthermore, in order to obtain the pathophysiology and physiological / biochemical knowledge of arrhythmia, measurement of the heart and surrounding size, calculation of cardiac output, confirmation of cardiovascular circulation status by angiography, histology Analysis, gene expression analysis, protein expression analysis, etc. may be performed together.

  In another aspect of the invention, a method is provided for assessing the likelihood that a test substance will induce torsades de pointes ventricular tachycardia in humans. In this method, a zebrafish fertilized egg within 6 days after fertilization is bred in the presence of the test substance, is bred in the absence of the test substance after ventricular arrest occurs, and then the ventricular pulsation is restored. Later, an electrocardiogram of zebrafish is measured to determine whether or not a torsade de pointes ventricular tachycardia is indicated. According to the method of the present invention, the risk that a drug induces arrhythmic side effects can be conveniently and easily tested.

  Furthermore, the model animal of torsade de pointes ventricular tachycardia obtained by the method of the present invention can be used to identify candidate substances for therapeutic agents for arrhythmia. In this method, the test substance is contacted with a zebrafish exhibiting a torsade de pointes ventricular tachycardia produced by the method of the present invention to determine whether the test substance changes the symptoms of arrhythmia in the zebrafish. To do. The test substance may be dissolved in the breeding water if it is water-soluble, and may be suspended in the breeding water in the form of a complex or emulsion with an appropriate surfactant if it is water-insoluble. Alternatively, it may be administered orally by mixing with zebrafish food, or parenterally by injection or the like. The test substance may be pre-administered before the zebrafish is exposed to the QT prolonging agent, may be administered simultaneously when exposed to the QT prolonging agent, or in the absence of the QT prolonging agent after ventricular arrest. You may administer when raising a zebrafish. Identify test substances that reduce or alleviate arrhythmia symptoms as candidates for arrhythmia drugs by measuring and comparing heart rate and electrocardiogram between the atrium and ventricle with and without test substance administration Can do.

  According to the present invention, a torsade de pointes type ventricular tachycardia model animal can be obtained by a simple method. Moreover, it can test by a simple method about the possibility that a test substance will cause a torsade de pointes type ventricular tachycardia.

  EXAMPLES The present invention will be described below in more detail with reference to examples, but the present invention is not limited to these examples.

Example 1. Ventricular arrest and recovery by astemizole administration The method of raising adult zebrafish (Tuebingen) was according to THE ZEBRAFISH BOOK (by Monte Westerfield, Institute of Neuroscience, University of Oregon) (URL: http://www.shigen.nig.ac .jp: 6070 / zf_info / zfbook / zfbk.html). On the day before the test, one male and one female were placed in a water tank, placed in a dark room, and lit eggs were promoted by lighting in the early morning, and fertilized eggs were collected. 150 fertilized eggs are divided into 3 groups (washed group, non-washed group, control group), and the breeding water (calcium chloride 0.1g, sodium chloride 3.5g, potassium chloride 0.05g, sodium bicarbonate 0.2) from immediately after fertilization to 3 days g / 1L). The density was kept by changing the breeding water every day in a constant temperature bath at 28 ° C. so that the amount of water per egg did not fall below 0.25 mL. Fertilized eggs hatched 2-3 days after fertilization. On the third day, astemizole (final concentration 0.5 μM or 1 μM) was added to the breeding water, and the breeding was continued. The control group was raised in breeding water not containing astemizole. On the 4th day (4dpf), the heart rate of the atrium (Atrium) and the ventricle (Ventricle) was measured for 15 seconds each. After the measurement, the non-washing group (ATZ) was reared with breeding water containing the same concentration of astemizole, and the washing group (ATZ wash (+)) was reared with breeding water not containing astemizole. The heart rate was measured again on the 5th day (5 dpf).

  As a result, it was found that larvae bred in the presence of astemizole had decreased atrial and ventricular pulsations on the 4th day, but the ventricular pulsations were greatly decreased and AV block was generated (Fig. 1). In addition, it was found that ventricular pulsation was recovered when raised in the absence of astemizole from the 4th day. On the other hand, in the non-washing group that continued breeding in the presence of astemizole, ventricular pulsation remained stopped.

Example 2 Preparation of torsade de pointes model by administration of astemizole In the same manner as in Example 1, 100 fertilized eggs were divided into two groups (test group and control group) and reared. On the fifth day after fertilization, the fry was transferred to a petri dish and reared in breeding water containing astemizole (final concentration 1 μM) for 2 days. Egg Water was used as the solvent. Two days after drug administration (7th day after fertilization), ventricular pulsation stopped, and swelling of the heart was observed. The ventricular pulsation was restored after changing to breeding water without astemizole and breeding for two more days. At this time, electrocardiograms were measured and many individuals showed prolongation between QTs. One individual also measured polymorphic ventricular tachycardia, which is a typical waveform for torsades de pointes ventricular tachycardia, with prolonged QT and altered QRS waveform (Fig. 2: (A) control. Group, (B) administration group). That is, by breeding a zebrafish fertilized egg in the presence of astemizole according to the present invention, a model animal exhibiting QT prolongation or torsade de pointes ventricular tachycardia could be obtained.

  The zebrafish exhibiting the torsade de pointes ventricular tachycardia of the present invention is useful as a model animal for studying the pathophysiology of arrhythmias and for research and development of arrhythmia therapeutics and preventives.

FIG. 1 shows the atrial and ventricular heart rates of zebrafish kept for 1 day in the presence of astemizole and then for 1 day in the absence of astemizole. FIG. 2 shows an electrocardiogram of zebrafish fed for 2 days in the presence of astemizole and then for 2 days in the absence of astemizole.

Claims (2)

A method for producing a zebrafish showing torsades de pointes type ventricular tachycardia, wherein a zebrafish fertilized egg within 6 days after fertilization is bred in the presence of a QT prolongation drug, and after the ventricular arrest occurs, the QT prolongation drug Breeding in the absence of. A method for evaluating the possibility that a test substance induces torsades de pointes ventricular tachycardia in humans. A zebrafish fertilized egg within 6 days after fertilization is raised in the presence of the test substance, and ventricular arrest is observed. After occurrence, rear in the absence of the test substance, and then measure the zebrafish electrocardiogram after ventricular pulsation is restored to determine if it exhibits torsade de pointes ventricular tachycardia A method characterized by that.

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