JP2018104307A - Pharmaceutical composition - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a pharmaceutical composition useful for treatment and prevention of arrhythmia and long QT syndrome.SOLUTION: A pharmaceutical composition contains l-cis-diltiazem or salt thereof. In a myocardial cell in which l-cis-diltiazem being an optical isomer of d-cis-diltiazem which is clinically used as application to hypertension or angina is differentiated and induced from an iPS cell which is derived from a patient of long QT syndrome having CALCN1C gene mutation, delayed inactivation process is significantly accelerated without generation of potential-dependent type L calcium channel suppression.SELECTED DRAWING: None

Description

  The present invention relates to a pharmaceutical composition useful for the treatment and prevention of arrhythmia and long QT syndrome.

  QT prolongation syndrome refers to electrocardiogram QT prolongation and T-wave abnormalities and causes severe ventricular arrhythmias such as special ventricular tachycardia and ventricular fibrillation called TdP (torsade de pointes). It is a syndrome that results in cerebral ischemic symptoms such as dizziness and fainting and sudden cardiac death. Long QT syndrome is divided into congenital and secondary, and congenitals include Romano-Ward syndrome and Jervell and Lange-Nielsen syndrome. Secondary QT prolongation syndrome is a syndrome in which QT prolongation occurs secondarily due to drugs and the like.

Timothy syndrome (long QT syndrome type 8, LQT8), which is included in congenital long QT syndrome, is a poor prognosis caused by mutations in the CACNA1C gene that encodes the α subunit of the L-type Ca ²⁺ channel. Yes, it is characterized by not only prolonged QT time and severe arrhythmia, but also systemic dysfunction such as syndactyly, immunodeficiency and autism.

  Many studies have been conducted on Timothy syndrome, and verapamil, mexiletine, and ranolazine have been reported as effective drugs for the treatment of Timothy syndrome. The effect is weak. In addition, in experiments using cardiomyocytes (CM) prepared from iPS cells (human induced pluripotent stem cells (hiPSC)) derived from patients with Timothy syndrome, roscovitine is the cause of prolonged QT time. It has been reported to improve the delay of L-type calcium channel inactivation (Non-Patent Documents 1-4). However, roscovitine is a drug developed as an anticancer drug and cannot be used for Timothy syndrome.

  In QT prolongation syndrome, there is a new disease group that can be called Timothy subtype, which is caused by CACNA1C gene mutation but does not have a phenotype other than the heart and has only QT prolongation and arrhythmia. It has become clear.

  Although heart failure has increased explosively in recent years due to aging, its prognosis is greatly influenced by the development of severe ventricular arrhythmias including ventricular fibrillation. In addition, mild QT prolongation is observed, but it is thought that there is a functional disorder of the ryanodine receptor channel involved in Ca release from the sarcoplasmic reticulum. In other words, the peak of the amount of calcium released into the cell via the ryanodine receptor channel decreases, and this delays the inactivation of the L-type calcium channel, resulting in the calcium channel in the second half of the action potential plateau phase. It is thought that calcium ions flowing into the cell increase through the action potential prolongation (extension of QT time on the electrocardiogram) and calcium overload.

  Diltiazem hydrochloride (Diltiazem hydrochloride, d-cis-Diltiazem hydrochloride) is a calcium channel blocker of benzothiazepine derivatives. By blocking the voltage-gated L-type calcium channel in a concentration-dependent manner, it exerts a vasodilatory effect and is indicated for hypertension and angina. On the other hand, it also suppresses calcium channels in the heart's stimulation conduction system and is used as an antiarrhythmic drug. Diltiazem has D-type and L-type as optical isomers, but the D-type is currently in clinical use.

Br. J. Pharmacol. 2007; 152, 386-395 J. Physiol. 2009; 587.3, 551-565 J. Biol. Chem. 2010; 285 (1), 43-53 Nature. 2011; 471 (7337), 230-234

  An object of the present invention is to provide a pharmaceutical composition useful for the treatment and prevention of arrhythmia and long QT syndrome.

  As a result of intensive studies to achieve the above-mentioned object, the present inventors have determined that i-cis-diltiazem, an optical mutant of d-cis-diltiazem, is an iPS derived from a patient with long QT syndrome having a CALCN1C gene mutation. In cardiomyocytes induced to differentiate from cells, it was found that the delayed inactivation process can be significantly promoted (improved) without causing voltage-dependent L-type calcium channel suppression (see FIG. 1). . In contrast, d-cis-diltiazem caused current blocking and had no effect on the delayed inactivation process.

  Based on these findings, the present invention has been completed through further studies, and provides the following pharmaceutical composition.

Item 1. A pharmaceutical composition comprising l-cis-diltiazem or a salt thereof.
Item 2. Item 2. The pharmaceutical composition according to Item 1, which is used for treatment and / or prevention of arrhythmia.
Item 3. Item 2. The pharmaceutical composition according to Item 1, which is used for treatment and / or prevention of ventricular arrhythmia.
Item 4. Item 2. The pharmaceutical composition according to Item 1, which is used for treatment and / or prevention of QT prolongation ventricular arrhythmia.
Item 5. Item 2. The pharmaceutical composition according to Item 1, which is used for treatment and / or prevention of long QT syndrome.

  The pharmaceutical composition of the present invention can significantly accelerate the delayed inactivation process of L-type calcium channels and has excellent properties of not suppressing peak current, resulting in improved QT prolongation Is possible. Therefore, the pharmaceutical composition of the present invention is expected to have a therapeutic and prophylactic effect on QT prolongation syndrome and arrhythmia caused by QT prolongation. Since it does not suppress the peak of Ca current, it has no so-called negative inotropic action and can be used for heart failure.

  Conventionally, since there is no drug that is decisively effective for the QT prolongation syndrome based on Ca overload, the pharmaceutical composition of the present invention can be a breakthrough therapeutic agent. Furthermore, the pharmaceutical composition of the present invention may be able to suppress the onset of severe arrhythmia in a pathological condition that causes Ca overload in cardiomyocytes such as heart failure and improve the prognosis thereof. Therefore, the pharmaceutical composition of the present invention is expected to be a more versatile drug.

It is a figure which shows the outline of the experiment in an Example. From the cells obtained from patients with Timothy syndrome (LQT8), iPS cells are prepared, differentiated into cardiomyocytes, and their functions are evaluated by electrophysiological methods and drugs that affect the inactivation of Ca channel currents Explored. It is an experimental result using barium as a current carrier. (A) A typical example of Ca channel current recording by whole cell type patch clamp method in cells derived from healthy subjects (control) and LQT8 patients. (B) A graph in which two current traces when depolarized to 0 mV are superimposed and displayed. This shows the effect of l-cis-diltiazem on LQT8-hiPSC-CM and is the result of a membrane potential fixation experiment using calcium as a current carrier. (A) Ca channel current recording by whole cell type patch clamp method in LQT8 patient-derived cells (right is 14 minutes after starting recording, upper trace is when l-cis-diltiazem (10 μM) is administered The bottom is a record without administration). (B) A graph in which two current traces before and after administration of l-cis-diltiazem (10 μM) when depolarized to 0 mV are superimposed and displayed. Since the current decreases with time (rundown phenomenon), the peaks are matched (and so on). (C) Two time constants of Ca channel current inactivation measured at each test potential and plotted. n = 9, ^* p <0.05 (D) A graph plotting r300 at each test potential (that is, the ratio of the Ca channel current level value after 300 ms depolarization to the peak current value). n = 9, ^** p <0.01 (E) is a graph showing the current-voltage relationship in which the peak current value at each test potential is plotted against the membrane potential. FIG. 2 is a graph showing activation and inactivation curves in a steady state with l-cis-diltiazem (10 μM) administered (n = 9), without administration (n = 5). The smooth line is drawn to fit the Boltzmann equation. n = 9 This shows the effect of l-cis-diltiazem on LQT8-hiPSC-CM, and is a result of a voltage fixation experiment using barium as a current carrier. (A) Ba channel current recording by whole cell type patch clamp method in LQT8 patient-derived cells (upper trace before l-cis-diltiazem (10 μM) administration, lower 14 minutes after administration). (B) A graph in which two current traces before and after l-cis-diltiazem administration when depolarized to 0 mV are superimposed and displayed. (C) A graph in which the time constant of inactivation of barium current through a Ca channel is measured at each test potential and plotted. n = 6, ^* p <0.05, ^** p <0.01 (D) Graph plotting r300 at each test potential (i.e., ratio of barium current value through Ca channel after 300 ms depolarization to peak current value) It is. n = 6, ^** p <0.01 (E) It is a graph which shows the inactivation gate of the barium current through the Ca channel in each test potential. The smooth line is drawn to fit the Boltzmann equation. n = 6, ^** p <0.01 It shows the effect of l-cis-diltiazem on LQT8-hiPSC-CM and is an experimental result of measuring action potentials from cardiomyocytes derived from LQT8 patients by current fixation method. (A) It is a graph which shows the action potential when depolarizing stimulation is given at 0.5 Hz, and shows a trace before and after administration of l-cis-diltiazem. ▼ indicates trigger pacing. (B) is a graph showing action potential duration (APD) before and after l-cis-diltiazem (10 μM) administration measured by 0.5 Hz pacing. n = 8, ^* p <0.01 (C) It is a graph which shows the parameter of the action potential before and after administration of l-cis-diltiazem (10 μM). MDA: Maximum resting membrane potential, APA: Action potential amplitude, n = 8 This shows the effect of d-cis-diltiazem on LQT8-hiPSC-CM, and is the result of a membrane potential fixation experiment using calcium as a current carrier. (A) Ca channel current recording by whole-cell patch clamp method in LQT8 patient-derived cells (right is 14 minutes after recording starts, upper trace is when d-cis-diltiazem (10 μM) is administered The bottom is a record without administration). (B) A graph in which two current traces before and after administration of d-cis-diltiazem (10 μM) when depolarized to 0 mV are superimposed and displayed. (C) A graph plotting r300 (that is, the ratio of the current value after 300 ms depolarization to the peak current value) at each test potential. n = 6 (D) is a graph showing the current-voltage relationship in which the peak current value at each test potential is plotted against the membrane potential. d-cis-diltiazem (10 μM) administered (n = 6), not administered (n = 5), ^* p <0.05, ^** p <0.01 This shows the effect of d-cis-diltiazem on LQT8-hiPSC-CM, and is a result of a voltage fixation experiment using barium as a current carrier. (A) Ba channel current recording by whole cell type patch clamp method in cells derived from LQT8 patients (upper trace before d-cis-diltiazem (10 μM) administration and lower 14 minutes after administration). (B) A graph in which two current traces before and after administration of d-cis-diltiazem (10 μM) when depolarized to 0 mV are superimposed and displayed. (C) A graph in which the time constant of inactivation of barium current through a Ca channel is measured at each test potential and plotted. n = 5 (D) A graph plotting r300 at each test potential (that is, the ratio of the barium current value after 300 ms depolarization to the peak current value). n = 5

  Hereinafter, the present invention will be described in detail.

  In the present specification, “comprise” includes the meaning of “essentially consist of” and the meaning of “consist of”.

  The pharmaceutical composition of the present invention comprises l-cis-diltiazem or a salt thereof.

  l-cis-Diltiazem has the chemical name: (2R, 3R)-(−)-cis-3-acetoxy-5- [2- (dimethylamino) ethyl] -2,3-dihydro It is 2- (4-methoxyphenyl) -1,5-benzothiazepin-4 (5H) -one and has a structure represented by the following formula.

  l-cis-diltiazem can be used in the free or salt state. Examples of the salt include salts with inorganic bases such as sodium, potassium, calcium, magnesium and aluminum; salts with organic bases such as methylamine, ethylamine and ethanolamine; and basic amino acids such as lysine, arginine and ornithine. Salts and ammonium salts. The salt may be an acid addition salt. Examples of the salt include mineral acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, nitric acid, phosphoric acid; formic acid, acetic acid, propionic acid, Organic acids such as oxalic acid, malonic acid, succinic acid, fumaric acid, maleic acid, lactic acid, malic acid, tartaric acid, citric acid, methanesulfonic acid and ethanesulfonic acid; acid addition with acidic amino acids such as aspartic acid and glutamic acid Salt. In addition, l-cis-diltiazem includes hydrates, solvates and polymorphs thereof.

  The content of l-cis-diltiazem in the pharmaceutical composition of the present invention is appropriately selected from the range of 0.001 to 100% by mass, preferably 0.01 to 99.9% by mass, more preferably 0.1 to 99% by mass in the total amount of the pharmaceutical composition. Can do.

  In addition to l-cis-diltiazem, the pharmaceutical composition of the present invention includes, if necessary, excipients, binders, disintegrants, lubricants, coloring agents, suspending agents, thickeners, Pharmaceutically acceptable components such as an oxidizing agent, an absorption accelerator, a pH regulator, a preservative, a preservative, a stabilizer, a surfactant, a sweetener, a corrigent, and a fragrance can be appropriately blended.

  Examples of the excipient include lactose, sucrose, purified sucrose, D-mannitol, D-sorbitol, corn starch, potato starch, dextrin, crystalline cellulose, sodium carboxymethylcellulose, light anhydrous silicic acid, talc, kaolin and the like. .

  Examples of the binder include pregelatinized starch, gelatin, gum arabic, methylcellulose, ethylcellulose, carboxymethylcellulose, sodium carboxymethylcellulose, crystalline cellulose, sucrose, purified sucrose, pullulan, hydroxypropylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone and the like. It is done.

  Examples of the disintegrant include lactose, sucrose, corn starch, potato starch, carboxymethyl cellulose, carboxymethyl cellulose calcium, croscarmellose sodium, carboxymethyl starch sodium, low-substituted hydroxypropyl cellulose, alginic acid, crospovidone and the like.

  Examples of the lubricant include stearic acid, magnesium stearate, calcium stearate, talc, polyethylene glycol and the like.

  Examples of the colorant include iron sesquioxide, yellow sesquioxide, brown iron oxide, black iron oxide, and titanium oxide.

  Examples of the suspending agent include polysorbate, polyethylene glycol, glycerin and the like.

  Examples of the preparation form of the pharmaceutical composition include tablets (including uncoated tablets, dragees, effervescent tablets, film-coated tablets, chewable tablets, troches, etc.), capsules, pills, powders (powder), granules, Forms such as fine granules, liquids, suspensions, emulsions, pastes, syrups, injections (including cases where they are prepared as liquids by mixing with infusions such as distilled water or amino acid infusions or electrolyte infusions at the time of use) Is mentioned. These preparations can be produced according to a conventional method.

  The administration method of the pharmaceutical composition of the present invention is not particularly limited, and can be performed by, for example, intraarterial administration, intravenous administration, buccal administration, rectal administration, enteral administration, transdermal administration, oral administration, and the like.

  The pharmaceutical composition of the present invention is administered to mammals including humans.

  The dosage of the pharmaceutical composition of the present invention can be appropriately determined according to various conditions such as the body weight, age, sex, and symptoms of the patient.

  The pharmaceutical composition of the present invention is useful for the prevention and / or treatment of arrhythmia. The “arrhythmia” in the present invention is preferably a ventricular arrhythmia, more preferably a QT prolongation ventricular arrhythmia. Preferred “arrhythmia” also includes ventricular arrhythmia due to abnormal calcium handling. Furthermore, the pharmaceutical composition of the present invention is also useful for the treatment and / or prevention of long QT syndrome.

  The pharmaceutical composition of the present invention can significantly accelerate the delayed inactivation process of L-type calcium channels and has excellent properties of not suppressing peak current, resulting in improved QT prolongation can do. Therefore, the pharmaceutical composition of the present invention is expected to have a therapeutic and prophylactic effect on QT prolongation syndrome and arrhythmia (particularly ventricular arrhythmia) caused by QT prolongation.

  In addition, since the pharmaceutical composition of the present invention does not suppress the peak of the Ca current, it has no so-called negative inotropic action (an action that reduces the contractile force of the myocardium) and can be used for heart failure.

  Conventionally, since there is no drug that is decisively effective for the QT prolongation syndrome based on Ca overload, the pharmaceutical composition of the present invention can be a breakthrough therapeutic agent. Furthermore, the pharmaceutical composition of the present invention may be able to suppress the onset of severe arrhythmia in a pathological condition that causes Ca overload in cardiomyocytes such as heart failure and improve the prognosis thereof. Therefore, the pharmaceutical composition of the present invention is expected to be a highly versatile drug.

  Currently used in clinical practice is the optical isomer d-cis-diltiazem, but as shown in the examples below, d-cis-diltiazem causes current blocking and delayed inactivation. The process was not affected at all. Thus, l-cis-diltiazem exhibits a completely different effect from the optical isomer d-cis-diltiazem.

  Examples are given below to illustrate the present invention in more detail. However, the present invention is not limited to these examples.

<Method of preparing hiPSC and induction of cardiomyocyte differentiation>
IPS cells were isolated and prepared from the peripheral blood of patients with CACNA1C-A582D mutation in LQT8. The details of the method were based on the following document (1). In addition, iPS cells derived from healthy subjects were also prepared as in the previous paper (2). Differentiation into cardiomyocytes was carried out using the embryooid bodies (EBs) formation method (3). After differentiation induction, the cells were isolated from the culture dish using collagenase and trypsin on the 20th day of culture and transferred to culture. See (3) for details.
(1) Okita K, Yamakawa T, Matsumura Y et al. An efficient nonviral method to
generate integration-free human-induced pluripotent stem cells from cord blood and peripheral blood cells.Stem Cells 2013; 31: 458-66.
(2) Kiyonaka S, Wakamori M, Miki T et al. RIM1 confers sustained activity and neurotransmitter vesicle anchoring to presynaptic Ca ²⁺ channels. Nat Neurosci 2007; 10: 691-701.
(3) Yang L, Soonpaa MH, Adler ED et al. Human cardiovascular progenitor cells develop from a KDR ⁺ embryonic-stem-cell-derived population. Nature 2008; 453: 524-8.

<Confirmation by gene search>
Genomes were isolated from iPS cell-derived cardiomyocytes of LQT8 patients and healthy individuals using the GenElute Mammalian Genomic DNA Miniprep kit (Sigma-Aldrich, St. 119 Louis, MO, USA). This was subjected to genetic testing by the classical PCR-Sanger method, and it was confirmed that the CACNA1C-A582D mutation was expressed in the former. Analysis was performed using 3130 Genetic Analyzer and Big Dye Terminator v3.1 (Thermo Fisher Scientific, Waltham, MA, USA).

<Electrophysiological recording method>
After induction of differentiation from iPS cells to cardiomyocytes, they were isolated using collagenase and trypsin, cultured on a cover glass coated with gelatin for a short period of time and subjected to electrophysiological experiments. Measurements were made using whole cell patch clamps and recordings were made using Axon 700B Multiclamp and Digidata 1440 digitizer hardware (Axon instruments, CA, USA). For data analysis, pClamp 10.4 software (Moleculr Devices, CA, US) was used. Myocardial action potentials were recorded in current clamp mode using cells 6-8 weeks after myocardial differentiation. l-cis-Diltiazem was purchased from Abcam (Cambridge, UK) and d-cis-Diltiazem was purchased from Sigma-Aldrich (St Louis, MO, USA). Solutions containing 10 μM l-cis-diltiazem or d-cis-diltiazem were applied 5 minutes prior to data acquisition. Details of the methodology followed literature (4).
(4) The latest patch clamp experiment technique. Yoshioka Shoten, Kyoto, 2011

<Statistical analysis>
All data are shown as mean ± standard error. Comparisons between two groups of normally distributed data were made using Student's t-test, and intracell comparisons were made using paired t-tests. Statistical significance was considered when p <0.05.

  FIG. 1 shows an outline of an experiment in the following test example. From the cells obtained from patients with Timothy syndrome (LQT8), iPS cells are prepared, differentiated into cardiomyocytes, and their functions are evaluated by electrophysiological methods and drugs that affect the inactivation of Ca channel currents Explored. The obtained experimental results are shown in FIGS. 2-7.

Test Example 1: Electrophysiological properties of LQT8-hiPSC-CM Fig. 2 shows the whole cell type patch clamp method in typical healthy control cells and LQT8 patient cells (LQT8-hiPSC-CM). A typical example of barium current recording through a Ca channel is shown. By using barium ions as carriers, it is possible to exclude the inactivation of the so-called Ca-dependent Ca current, and to observe the inactivation of the voltage-dependent Ca current. In FIG. 2B, two current traces when depolarized to 0 mV are superimposed, and from this information, LQT8-hiPSC-CM shows a peak barium current compared to cells derived from healthy subjects. It can be seen that the inactivation is delayed without decreasing.

Test Example 2: Effect of l-cis-diltiazem on LQT8-hiPSC-CM
The results of examining the action of l-cis-diltiazem are shown in FIGS. 3-5.

  FIG. 3 shows the results of a membrane potential fixation experiment using calcium as a current carrier. From FIG. 3B it is clear that l-cis-diltiazem improved Ca channel current inactivation. FIG. 3D shows that the r300 value is significantly decreased after l-cis-diltiazem administration. From FIG. 3E, although the peak current value tended to decrease before and after l-cis-diltiazem administration, it was not a significant decrease considering rundown. FIG. 3F also shows that l-cis-diltiazem does not affect steady-state activation and membrane potential dependence of the inactivated gate.

  FIG. 4 shows the results of a voltage fixing experiment using barium as a current carrier. FIG. 4B shows that l-cis-diltiazem tended to improve inactivation of L-type calcium channels as described above. FIG. 4D shows that the r300 value is significantly decreased after l-cis-diltiazem administration. FIG. 4E shows that L-type calcium channel inactivation occurs completely after l-cis-diltiazem administration on the depolarization side from 0 mV.

  From the results of FIGS. 3 and 4, it can be seen that l-cis-diltiazem significantly promoted current inactivation regardless of whether calcium or barium is the current carrier through the calcium channel protein. I understand. As a result, as shown in FIGS. 5A and 5B, the duration was significantly shortened. Also, from FIG. 5C, there was no significant change in the other action potential parameters.

  From the above results, l-cis-diltiazem means shortening the QT time (corresponding to action potential duration) in QT prolongation syndrome, more straightforwardly indicating that it helps treat the disease Is.

Test Example 3: Effect of d-cis-diltiazem on LQT8-hiPSC-CM
The results of examining the action of d-cis-diltiazem are shown in FIGS. 6-7.

  FIG. 6 shows the results of a membrane potential fixation experiment using calcium as a current carrier. FIG. 6B shows that d-cis-diltiazem has no effect on Ca current inactivation. From FIG. 6C, it can be seen that d-cis-diltiazem has no effect on the r300 value. FIG. 6D shows that d-cis-diltiazem suppressed the Ca channel current to a level of about one third.

  FIG. 7 shows the results of a voltage fixing experiment using barium as a current carrier. FIG. 7B shows that d-cis-diltiazem has no effect on the inactivation of L-type calcium channels in the same manner as described above. FIG. 7D shows that the r300 value did not change before and after the administration of d-cis-diltiazem.

  The clinically used d-cis-diltiazem is an optical isomer of l-cis-diltiazem, but in either case recording calcium or barium as the current carrier, as seen in FIGS. Although the peak current was significantly reduced (that is, it worked as a conventionally known Ca channel inhibitor), it had no effect on the current inactivation accompanying depolarization. Therefore, from the viewpoint of medicinal effect, it was found that the two optical isomers have completely different actions on the myocardial L-type calcium channel.

Claims (5)

  A pharmaceutical composition comprising l-cis-diltiazem or a salt thereof.   The pharmaceutical composition according to claim 1, which is used for treatment and / or prevention of arrhythmia.   The pharmaceutical composition according to claim 1, which is used for treatment and / or prevention of ventricular arrhythmia.   The pharmaceutical composition according to claim 1, which is used for treatment and / or prevention of QT prolongation ventricular arrhythmia.   The pharmaceutical composition according to claim 1, which is used for treatment and / or prevention of long QT syndrome.