WO2019131308A1 - TREATMENT FOR ARRHYTHMIA USING 9-β-D-ARABINOFURANOSYLHYPOXANTINE - Google Patents

Abstract

Provided is a novel therapeutic agent for arrhythmia. An anti-arrhythmic agent comprising 9-β-D-arabinofuranosylhypoxanthine, a pharmaceutically acceptable salt thereof or a solvate of the same.

Description

Arrhythmic treatment with 9-.BETA.-D-arabinofuranosyl hypoxanthine

The present invention relates to the treatment of arrhythmias with 9-β-D-arabinofuranosyl hypoxanthine (9-β-D-arabinofuranosylhypoxanthine (Ara-Hx)).

Arrhythmia causes heart failure, the onset of cerebral infarction, sudden death, etc., and greatly affects human life and quality of life (QOL), so the establishment of prevention and treatment of these is required. Atrial fibrillation, which is one of the clinically important arrhythmias, is said to affect more than 700,000 people in Japan (Non-patent document 1: Japan Cardiovascular Society JCS guidelines 2013) Not only symptoms, but a major cause of cerebral infarction. In addition, about 50% of deaths of relatively mild heart failure patients are reported to be sudden death (Non-patent document 2: O'Callaghan PA et. Al, European journal of heart failure 1999), but this important cause One of them is considered to be a severe ventricular arrhythmia. Although the development of radical treatment for arrhythmias due to ablation (percutaneous myocardial ablation) has been remarkable in recent years, there are still many dangerous arrhythmias that can not be treated by ablation. In addition, antiarrhythmic drugs need to be of great importance in the future as a means to prevent arrhythmia before onset.

One of the important mechanisms responsible for arrhythmias is excessive activation of sympathetic nerve signals. Noradrenaline, which is a sympathetic neurotransmitter, acts mainly through β-adrenergic receptors (β-AR) of cardiac muscle cells. The sympathetic nervous system exerts a physiological action important for maintenance and control of cardiac function, but at the same time induces an arrhythmia as an adverse effect or causes cardiomyocyte death to cause heart failure (Non-patent Document 3: Fujita, T., et al. Circ J 75: 1811-1818. 2011). A currently widely established therapeutic method for suppressing this adverse effect is the suppression of β-adrenergic receptor function by β-blockers (β-blockers). Although beta-blockers significantly improve the prognosis of heart failure patients, the main factor is believed to be the suppression of severe arrhythmias. However, since beta-blockers also inhibit important functions such as maintenance of basic cardiac function by sympathetic nerves, they cause serious side effects such as exacerbation of heart failure. This is a major problem as it often makes it difficult to start and continue treatment with β-blockers.

We have continued to develop drugs that selectively suppress the adverse effects of the sympathetic nervous system, which overcome these side effects. We focused on the downstream signaling system of beta-adrenergic receptors. When the β-adrenergic receptor is stimulated, it activates adenylate cyclase (AC), and the produced second messenger cAMP activates PKA and Epac to control cardiomyocyte function. There are nine subtypes of AC, and in particular, we have been deficient in type 5 AC (AC5) deficient mice (AC5KO) that are specifically expressed in the heart-specific manner (Non-patent document 4: Okumura, S. et al. Proc. Natl. Acad. Sci. USA 100, 9986-9990. 2003, Non-patent document 5: Okumura, S. et al. Circulation 118, 1776-1783. 2007), Epac deficient mice are prepared and their function in the heart (Non-patent document 6: Fujita, T. (co-first author), et al. J Clin Invest 124 2785-2801. 2014, non-patent document 7: Fujita, T. (co-first author), et al Biochem Biophys Res Commun 475, 1-7. 2016). The results show that AC5 and Epac selectively mediate the adverse effects of the sympathetic nervous system in the heart, and these molecules are safer than β-blockers, which also suppress physiological actions It is suggested that it could be a target of In fact, vidarabine (antiviral drug. Trade name: aracena) which was found to be an AC5 inhibitor by our screening (Non-patent document 8: Iwatsubo, K. et al. J. Biol. Chem. 279, 40938- 40945, 2004), it has been revealed that it is useful for treating arrhythmias and heart failure in animal experiments and can be used more safely than β-blockers (Non-patent Document 9: Am J Physiol Heart Circ Physiol 302: H2622-2628. 2012 Non-Patent Document 10: Fujita T (corresponding author) et al. Circ J 80, 2496-2505, 2016, Patent Document 1: Patent No. 6028983).

Patent No. 6028983

For many of the antiarrhythmic drugs developed so far (ion channel function, modulators of sympathetic nervous system signals, etc.), low safety is a major problem. In particular, many of them show depression of cardiac function as a side effect, and can not be used for patients with heart failure, which is the underlying disease of many severe arrhythmias. On the other hand, it often induces arrhythmias, requires careful attention to its use, and in some cases, it is often necessary to give up treatment with antiarrhythmic drugs. Therefore, the establishment of safer therapeutics with less of these side effects is awaited.
An object of the present invention is to provide a novel antiarrhythmic drug.

Arrhythmia is an important disease that causes heart failure, the onset of cerebral infarction, sudden death, etc., and greatly affects human lifespan and quality of life (QOL), but the drugs that have been developed so far include cardiac function suppression etc. Often have side effects such as Here, we focus on the signal transduction system downstream of the β-adrenoceptor, and cause arrhythmias to 9-β-D-arabinofuranosylhypoxanthine (9-β-D-arabinofuranosylhypoxanthine (Ara-Hx)) Animal) It was clarified in animal experiments that there is an inhibitory effect (Fig. 1).
Ara-Hx is a metabolite of vidarabine (which has been clinically used as an antiviral agent) which has been reported to have suppressive effect on arrhythmia so far (patent No. 6028983). Ara-Hx is considered to be safe to use, as it has extremely weak side effects, which are important side effects, compared with vidarabine and β-blocker, which is an existing antiarrhythmic drug. In addition, because it is more soluble in water than vidarabine and has a longer half-life in blood, it may be possible to increase the blood concentration at lower doses and may further reduce side effects. In addition, its use as an internal medicine that has not been realized with vidarabine is also promising.

The present invention provides an antiarrhythmic drug comprising 9-β-D-arabinofuranosyl hypoxanthine, a pharmaceutically acceptable salt or solvate thereof.
The present invention relates to the treatment and / or treatment of arrhythmia comprising administering to a subject a pharmaceutically effective amount of 9-β-D-arabinofuranosyl hypoxanthine, a pharmaceutically acceptable salt or solvate thereof. Or provide a preventive method.
The present invention provides 9-β-D-arabinofuranosyl hypoxanthine, a pharmaceutically acceptable salt or solvate thereof, for the treatment and / or prevention of arrhythmia.

The present invention enables arrhythmia treatment using 9-β-D-arabinofuranosyl hypoxanthine, a pharmaceutically acceptable salt or solvate thereof.
This specification includes the contents described in the specification and / or drawings of Japanese Patent Application No. 2017-250478, which is the basis of the priority of the present application.

FIG. 2 is a schematic view of heart disease treatment focusing on the signal transduction system downstream of β-adrenergic receptor. Examine the duration of atrial fibrillation (AF) in a mouse atrial fibrillation model. After intra-abdominal administration of Ara-Hx, atrial fibrillation was induced by transesophageal pacing. Atrial fibrillation duration was significantly shortened by Ara-Hx administration. Study duration of atrial fibrillation in mouse atrial fibrillation model. Atrial fibrillation was induced by transesophageal pacing, and Ara-Hx was intravenously administered during atrial fibrillation attack after onset of atrial fibrillation attack. Ara-Hx administration significantly reduced the duration of atrial fibrillation. Cytosolic Ca ²⁺ concentration was measured in cultured cardiomyocytes from mouse atria, and Ca ²⁺ leak from sarcoplasmic reticulum (sarcoplasmic reticulum (SR)) after stimulation with noradrenaline (NA) (left) and spontaneous Observed an increase in spontaneous Ca ²⁺ release (spontaneous SR Ca ²⁺ release (SCR)) (right figure). In the presence of Ara-Hx, the increase of Ca ²⁺ leak from SR and spontaneous Ca ²⁺ release was significantly suppressed. Intracytoplasmic Ca ²⁺ concentration was measured in cultured cardiac myocytes derived from mouse ventricle, and Ca ²⁺ leak from sarcoplasmic reticulum (SR) after isoproterenol (ISO) stimulation (left figure) , Observe the increase in spontaneous Ca ²⁺ release (spontaneous SR Ca ²⁺ release (SCR)) (right figure). In the presence of Ara-Hx, the increase of Ca ²⁺ leak from SR and spontaneous Ca ²⁺ release was significantly suppressed. Measurement of intracellular reactive oxygen species (ROS) in cultured rat cardiomyocytes. In the presence of Ara-Hx, the increase response of intracellular reactive oxygen species by isoproterenol stimulation was significantly suppressed. We evaluated adenylate cyclase activity in membrane proteins from mouse atrium and ventricular tissue. While vidarabine suppressed adenylate cyclase activity, Ara-Hx had no significant effect on adenylate cyclase activity. Measure the heart rate of the mouse. Metoprolol (β-blocker), vidarabine, and Ara-Hx were intravenously administered, and the heart rate was examined at 10 seconds after administration for 10 seconds. Metoprolol and vidarabine significantly reduced heart rate, but Ara-Hx had no significant effect on heart rate.

Hereinafter, embodiments of the present invention will be described in more detail.

The present invention provides an antiarrhythmic drug comprising 9-β-D-arabinofuranosyl hypoxanthine, a pharmaceutically acceptable salt or solvate thereof.

9-β-D-arabinofuranosyl hypoxanthine (9-β-D-arabinofuranosylhypoxanthine (Ara-Hx)) is represented by the following formula.

9-β-D-arabinofuranosyl hypoxanthine can be prepared by known methods, or commercially available products can be used.

Examples of pharmaceutically acceptable salts of 9-β-D-arabinofuranosyl hypoxanthine include salts such as sodium phosphate, sodium salt, potassium salt, hydrochloride, sulfate and the like. It is not limited to these.

Examples of pharmaceutically acceptable solvates of 9-β-D-arabinofuranosyl hypoxanthine include solvates such as water, methanol, ethanol, dimethylformamide, ethyl acetate, dimmertisulfoxide (DMSO) and the like. Although it can do, it is not limited to these.

It was confirmed that 9-β-D-arabinofuranosyl hypoxanthine shortens the atrial fibrillation duration and has an arrest effect on atrial fibrillation (Example described later). Thus, 9-β-D-arabinofuranosyl hypoxanthine, pharmaceutically acceptable salts and solvates thereof can be used as antiarrhythmic agents (atrial arrhythmia therapy, ventricular arrhythmia therapy). For example, tachyarrhythmia (atrial flutter, paroxysmal supraventricular tachycardia, ventricular tachycardia, ventricular fibrillation / ventricular flutter), bradycardia arrhythmia (sinus atrium block, atrioventricular block), extra systole (upper ventricle) Useful in the treatment and / or prevention of sexual, ventricular, etc. The present invention can be used for arrhythmia treatment at the time of cardiopulmonary resuscitation, heart failure treatment, diabetes treatment, cancer treatment and the like.

When 9-β-D-arabinofuranosyl hypoxanthine, or a pharmaceutically acceptable salt or solvate thereof is used as a medicament, a pharmaceutical preparation formulated by a conventional method (eg, injection, capsule, It can be administered to humans or animals as tablets, powders, granules and the like. For example, once or several times at a dose of about 5 to 50 mg / kg body weight per day, preferably about 5 to 15 mg / kg body weight per day, converted to the amount of active ingredient It is recommended to divide it into oral or parenteral (for example, nasal, rectal, transdermal, subcutaneous, intravenous, intramuscular, etc.), but the dosage and frequency of administration may be changed appropriately according to symptoms, age, administration method etc. It can. Carriers such as distilled water and physiological saline should be used when formulated into injections, and starches, lactose, sucrose, calcium carbonate etc. when formulated into capsules, tablets, powders, and granules. Excipients, starch paste, gum arabic, gelatin, sodium alginate, carboxymethylcellulose, hydroxypropyl cellulose and other binders, magnesium stearate, talc and other lubricants, starch, agar, crystalline cellulose, calcium carbonate, etc. It is preferable to use a disintegrant such as sodium hydrogen carbonate or sodium alginate. The content of active ingredient in the formulation can be varied between 1 and 99% by weight. For example, in the form of tablets, capsules, granules, powders and the like, it is preferable to contain 5 to 80% by weight of the active ingredient, and in the case of injection, to contain 1 to 10% by weight of the active ingredient It is preferable to

Hereinafter, the present invention will be specifically described based on examples, but the present invention is not limited to these examples.

[Example 1] Ara-Hx reduces the duration of atrial fibrillation in mice (preventive effect) (Fig. 2)
We examined the duration of atrial fibrillation in a mouse model of atrial fibrillation (AF). High-frequency electrical stimulation (burst pacing) was applied to the atrium using an electrode catheter inserted into the esophagus to induce atrial fibrillation in C57BL6 / N mice (JRC, Japan, 12-18 weeks old). We report that it is possible to induce long lasting atrial fibrillation by intraperitoneally administering noradrenaline (1.5 mg / kg) and performing this atrial burst pacing after enhancing sympathetic nerve activity. In this study (Fujita, T. (co-first author), et al. PLoS One 10 (2015) e0133664.), We examined this method. Intraperitoneal administration of Ara-Hx (Carbosynth, NA 03401) (7.5 mg / kg, 15 mg / kg) or control solution (control: physiological saline) and 30 minutes later intraperitoneal administration of noradrenaline (1.5 mg / kg) . Ten minutes later, atrial fibrillation attack was induced by atrial burst pacing, and the duration of atrial fibrillation was evaluated. Atrial fibrillation duration was significantly shortened by Ara-Hx administration (mean ± SEM, p <0.05, n = 6-7).

Example 2 Ara-Hx has the effect of stopping atrial fibrillation in mice (therapeutic effect) (FIG. 3)
We examined the therapeutic effect of Ara-Hx on atrial fibrillation treatment in atrial fibrillation (AF) model of mice (Japan SLC, Inc., Sakai, 12-18 weeks old). Long-lasting atrial fibrillation can be induced by intraperitoneal administration of noradrenaline (1.5 mg / kg) and increased sympathetic activity followed by atrial burst pacing (Fujita, T. (co- first author), et al. PLoS One 10 (2015) e0133664.). Atrial fibrillation attack was induced by this method, and Ara-Hx (5, 10, 20 mg / kg) or DMSO only (CTRL) was intravenously administered during the stroke one minute after the onset of atrial fibrillation. The duration of atrial fibrillation before return to sinus rhythm was measured, and the atrial fibrillation duration was significantly shortened by Ara-Hx administration (mean ± SEM, p <0.05, n = 4).

[Example 3] Ara-Hx suppresses Ca ²⁺ leak from sarcoplasmic reticulum of atrial cells and ventricular cells (FIGS. 4 and 5)
Intracellular Ca ²⁺ concentration was measured in cultured cardiomyocytes from mouse (Japan SLC Co., Ltd., rat, 12-18 weeks old) (Figure 4), Ca ²⁺ from sarcoplasmic reticulum (SR) The degree of leakage was evaluated by the frequency of changes in cytosolic Ca ²⁺ concentration (SR Ca ²⁺ leak) and spontaneous Ca ²⁺ release (SCR) in the presence of tetracaine. Leakage of Ca ²⁺ from this sarcoplasmic reticulum causes delayed after depolarization (DAD), and the resulting triggered activity is important for the duration of atrial fibrillation attack and the onset of ventricular arrhythmias. It is believed to be a mechanism (Chen PS et al. Circ Res 114: 1500-1515).
Stimulation of the sympathetic nervous system with noradrenaline (NA) enhances Ca ²⁺ leakage from sarcoplasmic reticulum, but this potentiation response is SR Ca 2 ⁺ leak in the presence of Ara-Hx (10 μM, 100 μM), spontaneously Any increase in Ca ²⁺ release frequency was significantly suppressed (mean ± SEM, p <0.05, n = 14-19). In CTRL, the measurement was performed in the solution, only the absence of the drug.
This indicates that Ara-Hx suppresses the mechanism of prolongation of atrial fibrillation attack by sympathetic nerve activation.
Also, in cultured cardiomyocytes derived from mouse (Japan SLC Co., Ltd., rabbit, 12-18 weeks old) (Fig. 5), Ara-Hx is a muscle by isoproterenol (ISO), a β-adrenoceptor stimulator. The leakage of Ca ²⁺ from the endoplasmic reticulum was significantly suppressed (mean ± SEM, p <0.05, n = 11-19). This action of Ara-Hx suggests that Ara-Hx may also be effective in suppressing ventricular arrhythmias. In CTRL, the measurement was performed in the solution, only the absence of the drug.

[Example 4] Ara-Hx suppresses the generation of reactive oxygen species in cardiomyocytes (FIG. 6)
Reactive oxygen species in cardiomyocytes are known to be an important mechanism for the onset of atrial fibrillation and ventricular arrhythmias (JY Youn et al. J Mol Cell Cardiol 62 (2013) 72-79, AD Hafstad et al. Basic Res Cardiol 108 (2013) 359.).
Reactive oxygen species in cultured cardiac myocytes derived from rat ventricle (Japan SLC Co., Ltd., Wistar rats, neonatal rats: cardiomyocytes isolated from the heart after 3 days of age) are used as cell-penetrating fluorescent probes, DCFH-DA (2 It was measured with ', 7'-Dichlorodihydrofluorescin diacetate). The concentration of intracellular reactive oxygen species was significantly elevated after 24 hours by β-adrenergic receptor stimulation by isoproterenol (ISO) (10 μM), but this elevation is significantly suppressed in the presence of Ara-Hx (Mean ± SEM, p <0.05, n = 11-19).
It has been suggested that the inhibitory action of Ara-Hx on reactive oxygen species is one of the mechanisms of arrhythmia suppression by Ara-Hx. In CTRL, the measurement was performed in the solution, only the absence of the drug.

Example 5 Ara-Hx has no direct inhibitory effect on AC (FIG. 7)
Since Ara-Hx is a metabolite of the AC5 inhibitor vidarabine, we examined the effect of Ara-Hx on adenylate cyclase (AC) activity.
Radioisotope-labeled cAMP ([ ¹²⁵ I]) in the presence of an AC stimulant, forskolin (50 μM), using membrane fractions from mouse atrium (atrium) tissue and ventricular (ventricle) tissue samples -cAMP) by radioimmunoassay.
In the presence of the AC5 inhibitor vidarabine (10 μM), AC activity in atrial and ventricular tissues was significantly suppressed (mean ± SEM, p <0.05, n = 4), but Ara-Hx (10 μM) ) Had no significant effect on adenylate cyclase activity.
From this, it became clear that Ara-Hx has very low direct inhibitory effect on AC. AC also plays an important role in physiological cardiac function control, and it has been suggested that Ara-Hx, which exerts antiarrhythmic action without affecting this AC activity, may have few side effects on cardiac function. In CTRL, the measurement was performed in the solution, only the absence of the drug.

[Example 6] Ara-Hx does not inhibit cardiac function (heart rate) (Fig. 8)
Heart rate is an important factor in defining cardiac function, and the effects of Ara-Hx on heart rate were examined. Mouse electrocardiogram was recorded under isoflurane anesthesia to measure heart rate. Metoprolol (β-adrenergic receptor blocker) (0.3 mg / kg), vidarabine (10 mg / kg), Ara-Hx (10 mg / kg) or DMSO only (CTRL) was administered intravenously and administered 30 After 10 seconds, the heart rate was evaluated. Metoprolol and vidarabine significantly reduced the heart rate (mean ± SEM, p <0.05, n = 5), while Ara-Hx had no significant effect on heart rate. The maximum dose of metoprolol for adults is 240 mg / day and vidarabine is 15 mg / kg / day. It was suggested that Ara-Hx had fewer side effects of cardiac depression compared to metoprolol and vidarabine.
All publications, patents and patent applications cited herein are incorporated herein by reference in their entirety.

The present invention is applicable to the treatment and / or prevention of arrhythmias.

Claims (3)

1. An antiarrhythmic drug comprising 9-β-D-arabinofuranosyl hypoxanthine, a pharmaceutically acceptable salt or solvate thereof.
2. A method of treating and / or preventing arrhythmia comprising administering to a subject a pharmaceutically effective amount of 9-β-D-arabinofuranosyl hypoxanthine, a pharmaceutically acceptable salt thereof or a solvate thereof.
3. 9-β-D-arabinofuranosyl hypoxanthine, a pharmaceutically acceptable salt or solvate thereof for the treatment and / or prevention of arrhythmia.

Images