Drugs for the treatment of inherited bradyarrhythmias

Abstract

The object of the present invention is to provide a therapeutic agent for hereditary bradyarrhythmia. The solution is a therapeutic agent for bradyarrhythmia, comprising the following compound (I) or compound (II) or a pharmacologically acceptable salt thereof as an active ingredient. (In the formula, Ph represents a phenyl group.)

Description

Technical Field

[0001] The present invention relates to a therapeutic drug for bradyarrhythmia. Background Art

[0002] Arrhythmia refers to a state in which the heart rate and the rhythm of the heart beat become irregular, showing various abnormalities on the electrocardiogram. Arrhythmia caused by abnormal heart rate can be classified into tachyarrhythmia and bradyarrhythmia. The heart rate of a person at rest is usually around 50 to 100 beats per minute. A heart rate lower than this is called bradycardia, and a heart rate higher than this is called tachycardia. Sick sinus syndrome (SSS), atrioventricular block (AV block), atrioventricular dissociation (AV dissociation), junctional rhythm, etc. can be cited as bradyarrhythmia.

[0003] The so-called sick sinus syndrome is a disease in which the pulse slows down mainly due to the decrease in the function of the sinus node, so that the function of the brain, heart, kidney, etc. is observed. In the electrocardiogram, it is classified into three types: 1) sinus bradycardia (heart rate 50 beats / minute or less), 2) sinus arrest or sinoatrial block, and 3) bradycardia-tachycardia syndrome. Clinically, symptoms such as Adam-Stokes syndrome, heart failure, and easy fatigability appear chronically. As a treatment method, oral drugs such as anticholinergics (atropine sulfate) and beta stimulants and intravenous injections are sometimes used to increase the number of spontaneous excitations of the sinus node. In the case where bradycardia does not improve even through their administration, or if the symptoms worsen if the drug administration is stopped, a pacemaker is sometimes used.

[0004] The sinoatrial node in the heart, which is the beginning of electrical stimulation, is a specialized part of the myocardium that has acquired autonomy and has specific protein expression. Among them, the most important for the function of the sinoatrial node is a group of cell membrane proteins called channels. Channels contribute to the formation of potential gradients inside and outside cells by allowing ions to pass through the cell membrane, and are related to the most important heart functions such as the propagation of electrical signals in not only the sinoatrial node but also all myocardial cells, and the subsequent contraction of the heart.

[0005] It has been known since ancient times that abnormalities in channel genes cause arrhythmias. Due to the recent advances in gene analysis technology, several channel genes have been identified as causative genes associated with arrhythmias.

[0006] In recent years, the present inventors have analyzed genes related to bradycardia in families with congenital bradycardia, and as a result, a new gene mutation of KCNJ3, one of the G protein-controlled potassium channels, has been identified among the channels that are important for the function of the sinoatrial node. For patients with an abnormally slow pulse phenotype, after obtaining informed consent from the patient and approval from the ethics committee of the facility, genetic analysis of the patient and his family was performed, and the results confirmed that the presence or absence of the bradycardia phenotype in the family is linked to the presence or absence of the gene mutation of KCNJ3. This mutation corresponds to a mutation (hereinafter sometimes referred to as "KCNJ3 N83H") in which the amino acid at the 83rd position from the N-terminus of the KCNJ3 protein, which is the gene product of KCNJ3, changes from asparagine (N) to histidine (H). Since the phenotype of KCNJ3 N83H presents bradycardia, it is suggested that the mutation involved in KCNJ3N83H of the KCNJ3 gene is dominant inheritance (for example, refer to Patent Document 1).

[0007] KCNJ3 protein, which is the gene product of KCNJ3, together with KCNJ5, constitutes the cardiac acetylcholine-activated potassium channel (KACh channel, or Kir3.1 / 3.4 channel) as a heterotetramer. If acetylcholine is released from the vagus nerve endings, the KACh channel opens via the activation of the M2 receptor, which is a muscarinic agonist acetylcholine receptor, and potassium ions flow out of the cell, thereby reducing the heart rate. In an experiment conducted by the two-electrode voltage clamp method using Xenopus oocytes, it was confirmed that before and after the addition of acetylcholine (ACh), the mutant (KCNJ3 N83H) KACh channel was 5 to 10 times more likely to flow current than the wild-type (KCNJ3 WT) KACh channel, and the mutant channel activity was greater.

[0008] The disease caused by the mutation of KCNJ3 has been redefined as one of the inherited bradyarrhythmias. The disease can also be called KACh channelopathy or Kir3.1 / 3.4 channelopathy. The disease is a rare arrhythmia in which the cardiac output required for the maintenance of biological functions is reduced due to bradycardia, which reduces physical activity / quality of life. In addition, as the disease progresses, there are serious risks such as low cardiac output heart failure, coma, and sudden death.

[0009] As described above, KACh channelopathy is a hereditary bradyarrhythmia caused by mutations in KCNJ3, and it is expected that KCNJ3 regulators (agonists or antagonists) will improve the condition. However, no specific compound targeting KACh channels and having a therapeutic effect on bradyarrhythmia has been reported so far.

[0010] In addition, it is unclear what regulatory factors are effective for treatment, and it is difficult to predict whether regulatory factors for human wild-type KCNJ3 will also be effective for "KCNJ3 N83H".

[0011] Prior art literature

[0012] Patent Literature

[0013] Patent Document 1: Japanese Patent Application Laid-Open No. 2010-136660 Summary of the invention

[0014] Problems to be solved by the invention

[0015] The present invention aims to provide a novel therapeutic drug for bradyarrhythmia.

[0016] Methods for solving problems

[0017] (3R, 4S) -7-hydroxymethyl-2,2,9-trimethyl-4- (phenylethylamino) -3,4-dihydro-2H-pyrano [2,3-g] quinolin-3-ol (hereinafter, also recorded as compound (I)) is a compound having a therapeutic effect on atrial fibrillation (see International Publication No. 2005 / 090357, etc.). The present inventors have found that compound (I) has an inhibitory effect on a human wild-type KACh channel (hereinafter sometimes referred to as "KCNJ3 WT KACh channel") composed of a human wild-type KCNJ3 protein, which is a gene product of human wild-type KCNJ3, and a human wild-type KCNJ5 protein, which is a gene product of human wild-type KCNJ5.

[0018] In addition, (3R, 4S)-6-amino-3, 4-dihydro-2, 2-dimethyl-7-nitro-4- (2-phenylethylamino) -2H-1-benzopyran-3-ol (hereinafter, also recorded as compound (II), compound (I) and (II) are also recorded as the compound of the present invention) has an atrial selective refractory period prolonging effect, and is reported to be effective against arrhythmias (see International Publication No. 01 / 21610, etc.). The present inventors have found that compound (II) also has an inhibitory effect on "KCNJ3 WT KACh channel".

[0019] As described above, the compounds of the present invention are known to have an anti-atrial fibrillation effect brought about by the inhibitory effect of the KCNJ3 WT KACh channel. However, in tachycardia diseases, especially high-speed atrial fibrillation, and hereditary bradyarrhythmias as slow diseases can also be said to be roughly the opposite symptoms. In addition, it is unclear whether the compounds of the present invention show an inhibitory effect on the mutant KACh channel (hereinafter sometimes referred to as "KCNJ3 N83HKACh channel") composed of KCNJ3N83H protein and human wild-type KCNJ5 protein. Therefore, it is difficult to predict whether the compounds of the present invention will become a therapeutic agent for hereditary bradyarrhythmias.

[0020] However, as a result of intensive studies, the present inventors surprisingly found that the compound of the present invention exhibits a stronger inhibitory effect in the "KCNJ3 N83H KACh channel" than in the "KCNJ3WT KACh channel".

[0021] Furthermore, the inventors have conducted research and have found that KACh channelopathy presents various symptoms in addition to sinus bradycardia, such as sinus arrest, sinoatrial block, and atrioventricular block. In the conventional treatment of arrhythmias, no existing drug is known that is effective in all of these various symptoms with a single dose.

[0022] However, the present inventors conducted intensive studies and surprisingly found that the compound of the present invention showed a high inhibitory effect on all symptoms of sinus bradycardia, sinus arrest, sinoatrial block, and atrioventricular block derived from the "KCNJ3 N83H KACh channel" in a zebrafish model, and it was clarified that the compound of the present invention can become the first drug for hereditary bradyarrhythmias (KACh ion channel diseases) caused by mutations in the KACh channel gene.

[0023] Furthermore, the present inventors conducted studies and found that the compound of the present invention also increased the heart rate in a wild-type zebrafish model, that is, it was confirmed that the compound of the present invention is also effective for bradycardia that is not dependent on KACh channel mutation.

[0024] Furthermore, it was confirmed that the compound of the present invention is also effective against arrhythmias caused by mutations in the KCNJ5 protein, particularly bradyarrhythmias caused by mutation of the 101st amino acid from the N-terminus of the KCNJ5 protein from tryptophan (W) to cysteine ​​(C).

[0025] Therefore, the present invention is described as follows. (1)

[0027] A therapeutic agent for bradyarrhythmia, comprising the following compound (I) or compound (II) or a pharmacologically acceptable salt thereof as an active ingredient.

[0028]

[0029]

[0030] (In the formula, Ph represents a phenyl group.) (2)

[0032] The therapeutic agent according to (1), wherein the bradyarrhythmia is a hereditary bradyarrhythmia. (3)

[0034] The therapeutic agent according to (1), wherein the bradyarrhythmia is any one of sinus bradycardia, sinus arrest, sinoatrial block and atrioventricular block. (4)

[0036] The therapeutic agent according to (1), wherein the bradyarrhythmia is any one of sinus bradycardia, sinus arrest, sinoatrial block and atrioventricular block, and is a hereditary bradyarrhythmia. (5)

[0038] The therapeutic agent according to any one of (1) to (4), comprising the following compound (I) or a pharmacologically acceptable salt thereof as an active ingredient.

[0039]

[0040] (In the formula, Ph represents a phenyl group.) (6)

[0042] The therapeutic agent according to any one of (1) to (4), comprising the following compound (II) or a pharmacologically acceptable salt thereof as an active ingredient.

[0043]

[0044] (In the formula, Ph represents a phenyl group.)

[0045] (7) The therapeutic agent according to any one of (1) to (6), wherein the bradyarrhythmia is an arrhythmia caused by a mutation of the 83rd amino acid from the N-terminus of the KCNJ3 protein from asparagine (N) to histidine (H).

[0046] (8) The therapeutic agent according to any one of (1) to (6), wherein the bradyarrhythmia is an arrhythmia caused by a mutation of the 101st amino acid from the N-terminus of the KCNJ5 protein from tryptophan (W) to cysteine ​​(C).

[0047] Effects of the Invention

[0048] The present invention provides a therapeutic agent for bradyarrhythmia. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 The graph shows changes in current amplitude of KCNJ3 WT KACh channel (Kir3.1-WT) and KCNJ3 N83H KACh channel (Kir3.1-N83H) expressed in Xenopus oocytes caused by compound (II) when detected using the two-electrode voltage clamp method.

[0050] Figure 2 This graph shows the rate of change (%) of the logarithm of the current amplitude of KCNJ3 WT KACh channel (WT) and KCNJ3 N83H KACh channel (N83H) expressed in Xenopus oocytes relative to the concentration of compound (II) (evaluated by the current amount at a membrane potential of +20 mV) when detected using the two-electrode voltage clamp method.

[0051] Figure 3 A gene construct (Kir3.1-WT) for producing transgenic zebrafish, comprising a gene in which three genes, human KCNJ3 WT, human KCNJ5 and mCherry, are linked by a 2A peptide; and a gene construct (Kir3.1-N83H) comprising a gene in which three genes, human KCNJ3 N83H, human KCNJ5 and mCherry, are linked by a 2A peptide.

[0052] Figure 4 These are photographs showing the expression levels of GFP and mCherry in the hearts of second-day-old KCNJ3 WT zebrafish and KCNJ3 N83H mutant zebrafish larvae expressing GFP in the ventricle and GFP and mCherry in the atrium, observed using a fluorescence microscope.

[0053] Figure 5 This is a graph obtained by performing a detailed analysis of the heart rate and pulsation rhythm using analysis software based on the animation of the pulsation of the venous sinus, atrium, and ventricle in each zebrafish before and after the treatment with compound (II) to the KCNJ3 WT zebrafish and the KCNJ3 N83H mutant zebrafish.

[0054] Figure 6 This is a graph comparing the heart rates before and after the action of compound (II) in KCNJ3 WT zebrafish and KCNJ3 N83H mutant zebrafish.

[0055] Figure 7This graph shows the current amounts at various membrane potentials measured using the two-electrode voltage clamp method when compound (I) at three doses of 0.01 μM, 0.1 μM, and 1 μM acted on KCNJ3 WT KACh channel (wild-type KACh channel) and KCNJ3 N83H KACh channel (N83H mutant channel) expressed in Xenopus oocytes.

[0056] Figure 8 This figure shows a detailed analysis of the heart rate and pulsation rhythm using analysis software based on animations of the pulsations of the venous sinus, atrium, and ventricle before and 1 hour after the treatment of 100 nM of compound (I) with KCNJ3 N83H mutant zebrafish that developed sinus arrest and atrioventricular block.

[0057] Fig. 9 This figure shows a detailed analysis of the heart rate and pulsation rhythm using analysis software based on animations of the pulsations of the venous sinus, atria, and ventricles before and 1 hour after the treatment of 100 nM Tertiapin-Q with KCNJ3 N83H mutant zebrafish in sinus arrest.

[0058] Fig.10 This graph shows the current amounts at various membrane potentials measured using the two-electrode voltage clamp method when compound (I) at three doses of 0.01 μM, 0.1 μM, and 1 μM acted on KCNJ5 WT / W101C KACh channels (W101C heterozygous mutant KACh channels) expressed in Xenopus oocytes.

[0059] Fig.11 This is a graph showing the inhibition rate of the current amount when compound (I) at three doses of 0.01 μM, 0.1 μM and 1 μM acts on KCNJ5 WT / W101C KACh channel (W101C heterozygous mutant KACh channel) expressed in Xenopus oocytes at two membrane potentials of -70 mV and +30 mV. DETAILED DESCRIPTION

[0060] The so-called hereditary bradyarrhythmia is a hereditary and slow arrhythmia found in a family with congenital bradycardia. The inventors have confirmed the linkage between the hereditary bradyarrhythmia of this family and the gene mutation of KCNJ3. More specifically, it has been confirmed that the mutation is a mutation from asparagine (N) to histidine (H) at the 83rd amino acid from the N-terminus of the KCNJ3 protein, which is the gene product of KCNJ3. KACh ion channel disease or Kir3.1 / 3.4 ion channel disease is understood to be a new symptom of hereditary and slow arrhythmia.

[0061] Therefore, the so-called hereditary bradyarrhythmia in the present invention is understood to be the diseases described below.

[0062] (1) Bradyarrhythmias based on KCNJ3 mutations.

[0063] (2) Bradyarrhythmia caused by a mutation in the 83rd amino acid from the N-terminus of the KCNJ3 protein.

[0064] (3) Bradyarrhythmia caused by mutation of the 83rd amino acid from the N-terminus of the KCNJ3 protein from asparagine (N) to histidine (H).

[0065] (4) The disease according to any one of (1) to (3), wherein the bradyarrhythmia is sinus bradycardia, sinus arrest, sinoatrial block or atrioventricular block.

[0066] In the present invention, sinus bradycardia is a condition in which the heart rate slows down despite a normal and regular beat rhythm among the symptoms of arrhythmia. Sinus bradycardia is generally understood as a condition in which the heart rate decreases to less than 50 beats per minute. Sinus bradycardia is understood as a condition of Group I based on Rubenstein's classification among sick sinus syndrome.

[0067] In the present invention, sinus arrest refers to a condition in which no electrical signal is generated from the sinoatrial node and no atrial excitation occurs.

[0068] In the present invention, sinoatrial block refers to a condition in which atrial excitation does not occur due to a disorder in the conduction of excitation between the sinoatrial node and the atrium.

[0069] In the present invention, atrioventricular block refers to a condition in which ventricular excitation does not occur due to a disorder in the conduction of excitation between the atria and ventricles. Atrioventricular block includes the following types of disease classification.

[0070] (1) First degree atrioventricular block

[0071] A condition in which the conduction time between the atria and ventricles is prolonged. (Maintaining the conduction of excitement)

[0072] (2) Second degree atrioventricular block: A sudden interruption of atrium-ventricle conduction. There are two types.

[0073] (2-1) Wenckebach type: The conduction between the atria and the ventricles is gradually prolonged, and then the excitation of the ventricles is lost.

[0074] (2-2) Mobitz type II: The conduction between atria and ventricles is not prolonged but the excitation of the ventricles is suddenly lost.

[0075] (2-3) High degree atrioventricular block: Atrioventricular conduction is only rarely observed.

[0076] (3) Third degree atrioventricular block: The conduction between the atria and ventricles is completely interrupted.

[0077] KCNJ3 is one of human genes, and the KCNJ3 protein, which is the gene product of KCNJ3, constitutes a cardiac acetylcholine-activated potassium channel (KACh channel, or Kir3.1 / 3.4 channel) as a heterotetramer together with the KCNJ5 protein.

[0078] In addition, the KCNJ3 protein is also called Kir3.1 or GIRK1, and the KCNJ5 protein is also called Kir3.4 or GIRK4.

[0079] In the present invention, KCNJ3 N83H means, as described above, that the 83rd amino acid from the N-terminus of the KCNJ3 protein, which is the gene product of KCNJ3, is changed from asparagine (N) to histidine (H).

[0080] The patient to whom the therapeutic agent of the present invention is applied is not limited as long as the therapeutic agent is effective. Specifically, the patient to whom the therapeutic agent of the present invention is applied is any of the following aspects.

[0081] (1) Patients who are at risk of hereditary bradyarrhythmia.

[0082] (2) Patients diagnosed with hereditary bradyarrhythmias.

[0083] (3) Patients with KCNJ3 gene mutations.

[0084] (4) Patients having a mutation in the 83rd amino acid from the N-terminus of the KCNJ3 protein.

[0085] (5) Patients in which the 83rd amino acid from the N-terminus of the KCNJ3 protein is changed from asparagine (N) to histidine (H).

[0086] (6) Patients with KCNJ5 gene mutations

[0087] (7) Patients with hereditary and slow arrhythmias.

[0088] (8) Patients with bradyarrhythmia.

[0089] (9) Patients with cardiac arrhythmia.

[0090] The disease that is the target of the therapeutic agent of the present invention is not limited as long as the therapeutic agent is effective. The symptoms that are the target of the therapeutic agent of the present invention may include the following.

[0091] (1) Arrhythmia, (2) bradyarrhythmia, (3) sinus bradycardia, (4) sinus arrest, (5) sinoatrial block, (6) atrioventricular block, (7) sinus node dysfunction, and (8) chronic heart failure.

[0092] As described above, genetic analysis of patients with an abnormally slow pulse phenotype and their families was performed, and it was confirmed that the presence or absence of the bradycardia phenotype in the family was linked to the presence or absence of the KCNJ3 gene mutation.

[0093] The inventors have also identified rare mutations other than N83H in KCNJ3 and KCNJ5 that may also be associated with arrhythmia conditions.

[0094] The mutations other than N83H are (1) KCNJ3 F85L, (2) KCNJ3 N496H, (3) KCNJ5 D262G, (4) KCNJ5 V303I, and (5) KCNJ5 G387R.

[0095] In addition to the above-mentioned patent document 1 (Japanese Patent Laid-Open No. 2010-136660), after the priority date of the present invention, an example of hereditary bradyarrhythmia caused by mutations in the GIRK channel, particularly mutations in KCNJ5, was reported (see Circ Genom Precis Med. 2019; 12: e002238 published on January 15, 2019). As described above, arrhythmias caused by mutations in KCNJ5 are also the subject of treatment of the present invention.

[0096] As another embodiment, the mutation other than N83H is (6) KCNJ5 W101C.

[0097] The compounds of the present invention described in this specification are likely to have therapeutic effects on arrhythmias based on the mutations (1) to (6) above.

[0098] Furthermore, the effects of the compounds of the present invention on the above mutations (1) to (6) and the effects on the KCNJ3 N83H mutation may be synergistic, additive, or may have other effects.

[0099] Therefore, diseases that are targeted by the therapeutic agent of the present invention include the following diseases in addition to the bradyarrhythmia caused by the N83H mutation of KCNJ3 (hereinafter referred to as (A)).

[0100] (A1) Arrhythmia caused by mutation of the 85th amino acid from the N-terminus of the KCNJ3 protein from (F) to (L).

[0101] (A2) Arrhythmia caused by mutation of the 496th amino acid from the N-terminus of the KCNJ3 protein from (N) to (H).

[0102] (B) Cardiac arrhythmias based on KCNJ5 mutations.

[0103] (B1) Arrhythmia caused by mutation of the 262nd amino acid from the N-terminus of the KCNJ5 protein from (D) to (G).

[0104] (B2) Arrhythmia caused by mutation of the 303rd amino acid from the N-terminus of the KCNJ5 protein from (Y) to (I).

[0105] (B3) Arrhythmia caused by mutation of the 387th amino acid from the N-terminus of the KCNJ5 protein from (G) to (R).

[0106] Among these five diseases, more important diseases are (A2) and (B1), and atrial fibrillation is particularly assumed as the arrhythmia.

[0107] As another embodiment, diseases that are targeted by the therapeutic agent of the present invention include the following diseases in addition to the bradyarrhythmia caused by the above-mentioned KCNJ3 N83H mutation.

[0108] (B4) Arrhythmia caused by mutation of the 101st amino acid from the N-terminus of the KCNJ5 protein from tryptophan (W) to cysteine ​​(C).

[0109] Disease (B4) is also important, and as this arrhythmia, bradyarrhythmia is assumed.

[0110] Therefore, the detailed regimen of the patient to be treated with the therapeutic agent of the present invention also includes the following.

[0111] (10) A patient in which the 101st amino acid from the N-terminus of the KCNJ5 protein is changed from tryptophan (W) to cysteine ​​(C).

[0112] In addition, for the sake of caution, the single-letter notation of the amino acids in the above description is supplemented as follows: F: phenylalanine, L: leucine, N: asparagine, H: histidine, D: aspartic acid, G: glycine, V: valine, I: isoleucine, R: arginine, W: tryptophan, C: cysteine.

[0113] The compound (I) contained in the therapeutic agent of the present invention is, as described above, (3R,4S)-7-hydroxymethyl-2,2,9-trimethyl-4-(phenethylamino)-3,4-dihydro-2H-pyrano[2,3-g]quinolin-3-ol.

[0114] As one embodiment, compound (I) may be a racemic mixture or a diastereomeric mixture containing (3R, 4S) form.

[0115] As another embodiment, the compound contained in the therapeutic agent of the present invention may be an analog of compound (I). Examples of such analogs include compounds described in International Publication No. 2005 / 090357.

[0116] The compound (II) contained in the therapeutic agent of the present invention is, as described above, (3R,4S)-6-amino-3,4-dihydro-2,2-dimethyl-7-nitro-4-(2-phenylethylamino)-2H-1-benzopyran-3-ol.

[0117] As one embodiment, compound (II) may be a racemic mixture or a diastereomeric mixture containing (3R, 4S) form.

[0118] As another embodiment, the compound included in the therapeutic agent of the present invention may be an analog of compound (II). Examples of such analogs include compounds described in International Publication No. 01 / 21610.

[0119] The compounds (I) and (II) contained in the therapeutic agent of the present invention may be in the form of pharmacologically acceptable salts.

[0120] Examples of the pharmacologically acceptable salt include hydrochloride, hydrobromide, sulfate, methanesulfonate, acetate, benzoate, tartrate, phosphate, lactate, maleate, fumarate, malate, gluconate, and salicylate.

[0121] Preferred salts of compound (I) are hydrochloride, maleate and methanesulfonate.

[0122] A preferred salt of compound (II) is hydrochloride.

[0123] The compound contained in the therapeutic agent of the present invention may be a crystal. As long as the purpose of the present invention is not impaired, the crystal may be in any crystalline form. As an example of a preferred crystalline form of compound (I), the crystalline form described in International Publication No. 2010 / 126138 may be cited. As one embodiment, the crystal is a crystal having characteristic peaks at diffraction angles 2θ=5.6, 8.2, 12.0, 14.7, 16.6, 16.9, 17.9, 18.4, 22.5, 24.5, 27.6 (± 0.2°) as a pattern of powder X-ray diffraction when Cu·Kα is used as an X-ray source. As another embodiment, the crystal is a crystal having any 3, 5, 7 or 9 peaks among the above 11 peaks. The identification of the crystalline form using powder X-ray diffraction is based on the technical common sense of those skilled in the art, for example, the records of the Japanese Pharmacopoeia. When X-rays other than Cu·Kα rays are used, the pattern of the diffraction angle is compared by conversion of the 2θ value based on the Bragg formula. The measurement error of the 2θ value is generally within the range of ±0.2°, but even if a small number of peaks having an error of 0.2° or more exist, this does not prevent a person skilled in the art from making a reasonable identification.

[0124] The compound (I) included in the therapeutic agent of the present invention can be manufactured by a known method. The so-called known method is, for example, International Publication No. 2005 / 090357 and International Publication No. 2010 / 126138. In addition, when manufacturing compound (I), International Publication No. 2007 / 105658, International Publication No. 2014 / 050613, International Publication No. 2014 / 051077, International Publication No. 2015 / 012271, etc. can be used as reference.

[0125] The compound (II) contained in the therapeutic agent of the present invention can be produced by a known method such as International Publication No. 01 / 21610.

[0126] The present invention provides a pharmaceutical or veterinary composition comprising an amount of a compound of the present invention that is effective for the above-mentioned treatments.

[0127] Examples of the administration form of the compound of the present invention include injections (subcutaneous, intravenous, intramuscular, intraperitoneal injections), parenteral administration using ointments, suppositories, aerosols, etc., and oral administration using tablets, capsules, granules, pills, syrups, soluble liquid preparations, emulsions, suspensions, etc.

[0128] The above-mentioned pharmaceutical or veterinary composition containing the compound of the present invention contains the compound of the present invention in an amount of about 0.01 to 99.5%, preferably about 0.1 to 30%, based on the weight of the total composition.

[0129] In addition to the compounds of the present invention or compositions containing the compounds, other pharmaceutically or veterinarily active compounds may also be contained.

[0130] Furthermore, these compositions may contain a plurality of compounds according to the invention.

[0131] The clinical dosage of the compound of the present invention varies depending on age, body weight, sensitivity of the patient, degree of symptoms, etc., but the effective dosage is usually 0.003 to 1.5 g per day for an adult, preferably about 0.01 to 0.6 g. However, dosages outside the above range may be used as required.

[0132] The compound of the present invention is formulated for administration by conventional pharmaceutical methods.

[0133] That is, tablets, capsules, granules, and pills for oral administration can be prepared using excipients such as saccharide, lactose, glucose, starch, and mannitol; binders such as hydroxypropylcellulose, syrup, gum arabic, gelatin, sorbitol, tragacanth, methylcellulose, and polyvinylpyrrolidone; disintegrants such as starch, carboxymethylcellulose or its calcium salt, microcrystalline cellulose, and polyethylene glycol; lubricants such as talc, magnesium or calcium stearate, and silicon dioxide; and lubricants such as sodium laurate and glycerin.

[0134] Injections, soluble liquid preparations, emulsions, suspensions, syrups and aerosols can be prepared using solvents for active ingredients such as water, ethanol, isopropanol, propylene glycol, 1,3-butylene glycol, polyethylene glycol; surfactants such as sorbitan fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene ethers of hydrogenated castor oil, and lecithin; suspending agents such as carboxymethyl sodium salt, cellulose derivatives such as methyl cellulose, natural rubbers such as tragacanth gum and gum arabic; preservatives such as esters of p-hydroxybenzoic acid, benzalkonium chloride, sorbates, and the like.

[0135] As ointments of percutaneous absorption preparations, for example, white vaseline, liquid paraffin, higher alcohols, polyethylene glycol ointments, hydrophilic ointments, aqueous gel bases and the like can be used.

[0136] Suppositories can be prepared using, for example, cocoa butter, polyethylene glycol, lanolin, fatty acid triglycerides, coconut oil, polysorbate, and the like.

[0137] Example

[0138] The present invention will be described in further detail below with reference to Examples, but the present invention is not limited to these Examples.

[0139] In addition, the terms used in the examples are supplemented below.

[0140] PCR: polymerase chain reaction

[0141] cRNA: complementary RNA

[0142] Addgene: the name of a non-profit plasmid library

[0143] DMSO: dimethyl sulfoxide

[0144] ND96 solution: the following aqueous solution

[0145] 96mM NaCl, 2mM KCl, 1.8mM CaCl2, 1mM MgCl2, 5mM HEPES, pH 7.4

[0146] Ba 2+ :Barium chloride aqueous solution (or other divalent barium salt aqueous solution)

[0147] mCherry: Red fluorescent protein (refer to Experimental Medicine Supplement Vol.35No.5, etc.)

[0148] GFP: Green fluorescent protein (see Japanese Journal of Pharmacology Vol.138(2011)No.1P13-17 etc.)

[0149] germline transmission

[0150] (Example 1)

[0151] In order to analyze the inhibitory effect of compound (II) on KCNJ3 N83H KACh channels, KACh channels were expressed by injecting cRNA of KCNJ3 N83H or wild-type KCNJ3 (KCNJ3WT) and cRNA of wild-type KCNJ5 into Xenopus oocytes, and the channel activity was confirmed by two-electrode voltage clamp method with electrodes inserted. By co-expressing with the β and γ subunits of the G protein, the total cellular current passing through the activated KACh channel can be directly measured.

[0152] The coding sequence of human KCNJ3 WT was amplified by PCR from a human KCNJ3 cDNA clone (Invitrogen) and subcloned in a pCS2+ vector (Addgene). The c.247A>C mutation of KCNJ3 N83H was introduced by site-directed mutagenesis using PCR. Similarly, the coding sequence of human KCNJ5 was amplified by PCR from a human KCNJ5 cDNA clone (Invitrogen) and subcloned in a pCS2+ vector. The cRNAs of KCNJ3 WT, KCNJ3 N83H, and KCNJ5 were synthesized by in vitro transcription from linearized cDNA using the mMESSAGE mMACHINE kit (Life Technologies). cRNA of KCNJ3N83H or KCNJ3 WT and cRNA of wild-type KCNJ5 were injected into Xenopus oocytes together with cRNA of β and γ subunits of G protein and incubated in ND96 solution at 18°C ​​for 48-96 hours.

[0153] The glass pipette electrode has a resistance of 0.3 to 1.0 MΩ when filled with a 3 M potassium chloride aqueous solution. The extracellular solution was a test bath solution containing 40 mM potassium chloride, 50 mM sodium chloride, 3 mM magnesium chloride, 0.15 mM niflumic acid, and 5 mM HEPES, and the pH was adjusted to 7.4 with potassium hydroxide.

[0154] The total cellular current through the expressed KACh channel was measured by the two-electrode voltage clamp method using a GeneClamp 500 amplifier (Molecular Devices). Compound (II) was dissolved in DMSO, and the perfusion solution was prepared in such a way that the final concentration became 0.001, 0.01, 0.03, 0.1, 1, and 10 μM. When the current amount became basically stable, the perfusion solution containing compound (II) was added from a low concentration, and then the current amount was recorded while replacing it with a high concentration perfusion solution in the same way. At the end of the recording, Ba was added 2+ (3 mM) and measured the endogenous leakage current. The pulse experiment method is shown in the legend of the figure. The measurement data were analyzed using Clampfit 10.2 (Molecular Devices). The IC50 value was calculated using GraphPad Prism version 5.00 (GraphPad Software).

[0155] Compound (II) also showed a concentration-dependent inhibitory effect on KCNJ3 N83H KACh channels, and the IC50 value was lower than that of KCNJ3 WT KACh channels (N83H mutant; 50±12nM (n=14), wild type; 230±60nM (n=13), p<0.01). Compound (II) showed a higher affinity for mutant KACh channels ( Figure 1 , 2 ).

[0156] (Example 2)

[0157] In order to analyze the inhibitory effect of compound (II) on KCNJ3 N83H KACh channels in vivo, a gene encoding KCNJ3N83H was integrated into fertilized eggs of zebrafish (Danio rerio) to produce transgenic zebrafish into which KCNJ3 mutation was introduced as a disease model animal. As a comparative example, a zebrafish expressing KCNJ3 WT was also produced in the same manner.

[0158] Transgenic zebrafish were produced by using the transposon transfer system using the transposon Tol2. Since the cardiac KACh channel is expressed in the sinoatrial node, atrioventricular node, and atrial muscle, a gene construct was produced in zebrafish in which three genes, human KCNJ3 WT or N83H, human KCNJ5, and mCherry, were linked with a 2A peptide under the control of the amhc (atrial myosin heavy chain) promoter that specifically expresses the target gene in the atrium. The mCherry gene allows the gene introduction of KCNJ3 WT or N83H and human KCNJ5 to be distinguished under a fluorescence microscope ( Figure 3 ). Next, the mRNA of the transferase encoded by Tol2 and the construct were simultaneously microinjected into fertilized eggs. The adult fish thus formed were mated with wild-type zebrafish, and the fish whose germline transmission could be confirmed by producing fry that were positive for mCherry in the atrium under a fluorescent microscope were set as F0 founders. The F0 founders were mated with wild-type zebrafish hspGFF3A (Proceedings of the National Academy of Sciences of the United States of America. 2008; 105: 1255-1260.) that expressed GFP in both the ventricle and atrium, thereby using F1 transgenic zebrafish fry (2 days after birth) that expressed GFP in the ventricle and GFP and mCherry in the atrium. Figure 4), and an analysis of heart rate and cardiac pulsation rhythm was performed. Specifically, zebrafish larvae were fixed with 0.5% low-melting-point agarose gel on a 35 mm diameter glass-bottomed culture dish, and the hearts of the larvae were observed with a fluorescence microscope in a 0.03% sea salt solution. The animation of the heart beat obtained with the attached camera of the fluorescence microscope was used to perform a detailed analysis of the heart rate and pulsation rhythm using the original M-mode analysis software (The Journal of clinical investigation. 2007; 117: 2812-2824.). The heart rate measured the pulsation of the venous sinus where the sinoatrial node is located. No difference in the expression level of mCherry in the atrium was confirmed between KCNJ3 WT zebrafish and KCNJ3 N83H mutant zebrafish, so it can be considered that the expression level of the KACh channel gene is the same in both. In KCNJ3 N83H mutant zebrafish, significant atrial enlargement and bradyarrhythmic phenotypes similar to those in human cases (sinus arrest, sinus bradycardia, sinoatrial block, atrioventricular block) were observed ( Figure 5 ). Next, the 0.03% sea salt solution was replaced with a 0.03% sea salt solution to which 100 nM of compound (II) was added, and the heart beats were analyzed in the same manner after 1 hour. Surprisingly, 100 nM of compound (II) showed an increase in heart rate and an improvement in bradyarrhythmia in KCNJ3 N83H mutant zebrafish ( Figure 5 , 6 ).

[0159] in addition, Figure 5 The scale of the horizontal axis in is 2 seconds.

[0160] In summary, it is suggested that compound (II) also has an inhibitory effect on KCNJ3 N83H KACh channels in vivo and can be used as a therapeutic drug for hereditary bradyarrhythmias caused by mutations in the KACh channel gene. Furthermore, compound (II) does not cause excessive tachycardia in KCNJ3 WT zebrafish, but leads to a moderate increase in heart rate, suggesting the possibility of showing effectiveness even for bradycardia that is not dependent on mutations in the KACh channel gene ( Figure 6 ).

[0161] (Example 3)

[0162] Regarding compound (I), the inhibitory effect on KCNJ3N83H KACh channels and the improvement effect on increased heart rate and bradyarrhythmia in mutant zebrafish were confirmed in the same manner as in Examples 1 and 2, namely, the two-electrode voltage clamp method in Xenopus oocytes and the experiment using a transgenic zebrafish model ( Figure 7 , 8 ).

[0163] Compound (I) had an inhibitory effect not only on KCNJ3 WT KACh channels, but also on KCNJ3 N83H KACh channels. However, its effect was stronger on N83H than on WT ( Figure 7 ).

[0164] Furthermore, compound (I) improved sinus arrest and atrioventricular block based on N83H mutation in experiments using transgenic zebrafish models ( Figure 8 ).

[0165] (Example 4)

[0166] In the same manner as in Example 1, the inhibitory effect of compound (I) on KCNJ5 WT / W101C KACh channel (W101C heterozygous mutant KACh channel) was analyzed.

[0167] To mimic the autosomal dominant form of heterozygosity, equimolar amounts of wild-type KCNJ5 (KCNJ5 WT) and KCNJ5 W101C cRNAs were injected into Xenopus oocytes together with wild-type KCNJ3 (KCNJ3WT) cRNAs to express W101C heterozygous mutant KACh channels, and channel activity was confirmed by the two-electrode voltage clamp method using inserted electrodes.

[0168] The above-mentioned human KCNJ5 cDNA clone (Invitrogen) was amplified by PCR and subcloned in the pCS2+ vector (Addgene). Using this as a template, the c.303G>C mutation that can obtain KCNJ5 W101C was introduced by site-directed mutagenesis using PCR. KCNJ5 WT, KCNJ5 W101C, and KCNJ3 cRNAs were synthesized by in vitro transcription from linearized cDNA using the mMESSAGE mMACHINE kit (Life Technologies). Equimolar amounts of KCNJ5 WT and KCNJ5 W101C cRNAs and wild-type KCNJ3 cRNAs were injected into Xenopus oocytes and incubated in ND96 solution at 18°C ​​for 48-96 hours.

[0169] The glass pipette electrode has a resistance of 0.3 to 1.0 MΩ when filled with a 3 M potassium chloride aqueous solution. The extracellular solution was a test bath solution containing 40 mM potassium chloride, 50 mM sodium chloride, 3 mM magnesium chloride, 0.15 mM niflumic acid, and 5 mM HEPES, and the pH was adjusted to 7.4 with potassium hydroxide.

[0170] The total cellular current through the expressed KACh channel was measured by the two-electrode voltage clamp method using a GeneClamp 500 amplifier (Molecular Devices). Compound (I) was dissolved in DMSO and the perfusion solution was prepared in such a way that the final concentrations became 0.01, 0.1, and 1 μM. When the current flow rate became basically stable, the perfusion solution containing compound (I) was added from a low concentration, and then the current was recorded while replacing it with a high concentration perfusion solution in the same way. At the end of the recording, Ba was added 2+ The endogenous leakage current was measured by adding 1% dHO (3 mM). The pulse experiment method is shown in the legend of the figure.

[0171] Compound (I) also showed a concentration-dependent inhibitory effect on KCNJ5 WT / W101C KACh channel (W101C heterozygous mutant KACh channel) ( Fig.10 , 11 ).

[0172] (Comparative Example 1)

[0173] Tertiapin-Q is a peptide that has an inhibitory effect on KCNJ3 WT KACh channels (Synthesis of astable form of tertiapin: a high-affinity inhibitor for inward-rectifier K+ channels. Jin W, Lu Z. Biochemistry. 1999 Oct 26; 38(43): 14286-93.). Using Tertiapin-Q, which is an inhibitor of KCNJ3 WT KACh channels, the inhibitory effect on KCNJ3 N83H KACh channels was investigated.

[0174] Tertiapin-Q was tested using a transgenic zebrafish model in the same manner as in Example 2. Tertiapin-Q did not improve sinus arrest based on the N83H mutation ( Fig. 9 ).

[0175] Through this comparative experiment, it was determined that the therapeutic effect in the present invention is a unique effect of the compound of the present invention and is a significant effect.

[0176] Industrial Availability

[0177] According to the present invention, the compounds (I) and (II) of the present invention can be used as therapeutic agents for bradyarrhythmias.

Claims

1. Use of the following compound (I) or compound (II) or a pharmacologically acceptable salt thereof for preparing a medicament for use in the treatment of bradyarrhythmia, In the formula, Ph represents a phenyl group.

2. The use according to claim 1, wherein the bradyarrhythmia is a hereditary bradyarrhythmia.

3. The use according to claim 1, wherein the bradyarrhythmia is any one of sinus bradycardia, sinus arrest, sinoatrial block and atrioventricular block.

4. The use according to claim 1, wherein the bradyarrhythmia is any one of sinus bradycardia, sinus arrest, sinoatrial block and atrioventricular block, and is a hereditary bradyarrhythmia.

5. The use according to any one of claims 1 to 4, wherein the medicament uses the following compound (I) or a pharmacologically acceptable salt thereof as an active ingredient, In the formula, Ph represents a phenyl group.

6. The use according to any one of claims 1 to 4, wherein the medicament uses the following compound (II) or a pharmacologically acceptable salt thereof as an active ingredient, In the formula, Ph represents a phenyl group. 7 . The use according to any one of claims 1 to 4 , wherein the bradyarrhythmia is an arrhythmia caused by a mutation of the 83rd amino acid from the N-terminus of the KCNJ3 protein from asparagine N to histidine H. 8 . The use according to any one of claims 1 to 4 , wherein the bradyarrhythmia is an arrhythmia caused by a mutation of the 101st amino acid from the N-terminus of the KCNJ5 protein from tryptophan W to cysteine ​​C.

Images