EP1658312A1

EP1658312A1 - Modulators of the potassium channels twik-1, task-1, girk1, sk2 or pcn1, used to treat arrhythmia, coronary heart disease or hypertension

Info

Publication number: EP1658312A1
Application number: EP04740690A
Authority: EP
Inventors: Peter Ellinghaus; Klaus Münter
Original assignee: Bayer Healthcare AG
Current assignee: Bayer Pharma AG
Priority date: 2003-07-18
Filing date: 2004-07-06
Publication date: 2006-05-24
Also published as: WO2005016965A1; US20060183665A1; DE10332685A1

Abstract

The invention relates to the use of modulators of the potassium channels TWIK-1, TASK-1, GIRK1 SK2 and PCN1 in the production of a medicament for the treatment and/or prophylaxis of cardiac dysrhythmia (arrhythmia), coronary heart disease and hypertension.

Description

MODULATORS OF THE POTASSIUM CHANNELS T IK-1, TASK-1, GIRKl, SK2 OR PCNl FOR TREATING ARRHYTHMIA, CORONARY HEART DISEASES OR HIGH PRESSURE The invention relates to the use of potassium channel modulators for the manufacture of a medicament for the treatment of and / or preventive heart disease as well as high blood pressure or a combination of the diseases mentioned.

The cells of the sinus node in the right atrium of the heart have the function of a physiological pacemaker, since electrical excitation originates there at regular intervals. A change in membrane potential, which is determined by the concentration of different ions on both sides of a cell membrane, is responsible for the excitation conduction (Na ⁺ , K ⁺ ιmd Ca ²⁺ ). These ions pass through the cell membrane through ion-selective channels that consist of several subunits and together form a pore. During a heart action (systole), the heart muscle cell runs through an action potential, which is composed of phases 0-3 and in which all three types of ion channels mentioned above are involved. The action begins with a rapid depolarization (phase 0), in which primarily Na ⁺ channels are involved, followed by a transient, incomplete repolarization (phase 1) into the long-lasting one

Plateau phase (phase 2) passes and in which mainly Ca ²⁺ channels are involved. Phase 3 represents repolarization and is therefore responsible for restoring sleep. The KN outflow required for repolarization is mediated through potassium channels. During the entire action potential, the membrane is protected from a further depolarizing stimulus, it is refractory (1).

Arrhythmias either lead to disturbances in the formation of arousal, the conduction of the arousal or a combination of both. This can be caused by ischemia, inflammatory diseases of the heart muscle, but also intoxications or vegetative influences. Substances and processes that affect arousal formation or transmission are used therapeutically to treat arrhythmias. Substances that repolarize

Delaying the K ⁺ current and thereby lengthening the action potential duration and refractory period belong to the so-called class HI antiarrhythmics, of which A iodarone and Sotalol are currently approved in Germany (1).

Both substances, however, are not selective .. ahumkanalblockeπ So shows. Sotalol, in addition to blocking various K ⁺ channels (e.g. HERG), also has antagonistic properties for beta-adrenergic receptors, while amiodarone, in addition to HERG, also blocks the L-type Ca ²⁺ channel and Na ⁺ channel (1), ( 2). Just like the other classes of antiarrhythmics, the class ÜI potassium channel blockers have a considerable pro-arrhythmic potential, which is attributed to the simultaneous influence on the potassium channels in the ventricle and limits the clinical use. In this respect, the identification of potassium channels, which are preferably expressed in the atrium, as possible antiarrhythmic targets is of particular importance, since this could reduce the side effects, which can even lead to fatal rabbit fibrillation (3).

In addition to potassium channel blockers such as sotalol and amiodarone, anti-arrhythmic effects of potassium channel openers, e.g. B. for the ATP-dependent potassium channel (4).

In the present work, genes were identified using Affymetrix microarray technology, which are differentially expressed in the human heart between the left atrium and left ventricle (see FIG. 1). The differential expression of selected genes was verified using real-time PCR (TaqMan). It was shown that in all 6 patients examined the potassium channels TWIK-1 (5), TASK-1 (6), GIRKl (7), SK2 (8) and PCNl (9) were expressed much more strongly in the atrium than in the ventricle ( see Fig. 3).

The present invention therefore relates to the use of modulators of the aforementioned

Potassium channels for the manufacture of a medicament for the treatment and / or prophylaxis of the abovementioned diseases.

Potassium channel modulators in the sense of the present disclosure are substances which extend or shorten the opening time of the said potassium channels.

In the sense of the invention, modulators are all substances that change the biological

Cause channel activity. Particularly preferred modulators are nucleic acids including “locked nucleic acids”, “peptide nucleic acids” and “Spiegelmere”, proteins including antibodies and low molecular weight substances, very particularly preferred modulators are low molecular weight substances.

The invention relates to the use of modulators of the potassium channels TWIK-1, TASK-1,

GIRKl, SK2 or PCNl for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac arrhythmias (arrhythmias), coronary heart diseases or high blood pressure.

Furthermore, the invention relates to the use of modulators of the potassium channels TWIK-1, TASK-1, GIRKl, SK2 or PCNl with an IC ₅₀ of <1 μM, particularly preferably of <100 nM for the manufacture of a medicament for the treatment and / or prophylaxis of

Rhythm disorders (arrhythmias), coronary heart disease or high blood pressure. Another object of the invention is a method for screening test compounds for the identification of modulators of the potassium channels TWIK-1, TASK-1, GIRKl, SK2 or PCNl, which are suitable for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac arrhythmias (arrhythmias), coronary artery disease or high blood pressure. The invention also relates to a pharmaceutical composition containing one or more modulators of the potassium channels TWK-1, TASK-1, GIRKl, SK2 or PCNl for the treatment and / or prophylaxis of arrhythmias (arrhythmias), coronary heart diseases or high blood pressure.

The subject of the invention is also the use of modulators of the potassium channels TWIK-1, TASK-1, GIRKl, SK2 or PCNl for the regulation of the activity of the corresponding

Potassium channels in a living being, including humans, for the treatment and / or prophylaxis of heart rhythm disorders (arrhythmias), coronary heart disease or high blood pressure.

The invention also relates to modulators of the potassium channels TWIK-1, TASK-1, GIRKl, SK2 or PCNl for the treatment and / or prophylaxis of cardiac arrhythmias (arrhythmias), coronary

Heart disease or high blood pressure.

According to the invention is also the use of modulators of gene products, which are differentially expressed in the human heart between the left atrium and left ventricle, for the manufacture of a medicament for the treatment of arrhythmias, coronary heart diseases, high blood pressure and the consequences of atherosclerosis. Depending on the function of the

Enhanced expression in the ventricle may also be preferred (e.g. for the endothelin A receptor), the term differential gene expression is used here.

Another object of the invention is a method for screening test compounds for the identification of modulators of gene products which are differentially expressed in the human heart between the left atrium and left ventricle, which are suitable for the

Manufacture of a medicinal product for the treatment and / or prophylaxis of irregular heartbeat (arrhythmias), coronary artery disease or high blood pressure.

The invention also relates to a pharmaceutical composition, containing a modulator or modulators of gene products which are differentially expressed in the human heart between the left atrium and left ventricle, for treatment and / or

Prophylaxis of arrhythmias, coronary artery disease or high blood pressure. The invention furthermore relates to the use of modulators of gene products which are differentially expressed in the human heart between the left atrium and left ventricle for regulating the activity of the corresponding gene products in a living being, including humans, for the treatment and / or prophylaxis of cardiac arrhythmias (arrhythmias). , coronary artery disease or high blood pressure.

The invention also relates to modulators of gene products which are differentially expressed in the human heart between the left atrium and left ventricle, for the treatment and / or prophylaxis of arrhythmias (arrhythmias), coronary heart diseases or high blood pressure.

Substances that have a modulating effect on the activity of the channels mentioned can be identified using the assay described below (screening).

The anti-arrhythmic effect is tested in vivo using the animal experiment described below.

Description of the figures

Figure 1: Tabularly listed genes that were found to be differentially expressed between the atrium and ventricle in all 6 patients examined.

Figure 2: The Genbank Accession numbers of the genes verified by TaqMan-PCR and the primer / probe sequences used for this are listed in a table.

FIG. 3: The relative mRNA expression of the potassium channels TWIK-1, TASK-1 is shown.

GIRKl SK2 and PCNl in human hearts (left atrium [black] and left ventricle [white].

Figure 4: The relative protein expression of the potassium channel TASK-1 in human hearts is shown as the mean of all 6 patients, (left atrium [black] and left ventricle [white]. Examples

Example 1: Identification of differentially expressed genes between human ventricle and atrium

Small pieces (approx. 0.5 g) from the left ventricle or from the left atrium of explanted hearts were obtained with the consent of the donors from the Heart Center Halle (Prof. Morawietz). The total RNA from this was isolated after homogenization of the tissues using RNaesy columns (from Qiagen) according to the instructions. The description of each 10 μg total RNA in cDNA, its subsequent linear amplification and the hybridization of the biotinylated cRNA on human HG-U133A arrays was carried out according to the “Affymetrix User Guide” using Superscript-II (Gibco) and the “ High yield cRNA labeling kits (from Enzo).

The HG-U133A array basically allows the simultaneous mR A analysis of approx. 22,600 human genes. The arrays were evaluated using the software MAS 5.0 (Affymetrix) and Gene Spring 5.0 (Silicon Genetics). 1 summarizes the genes which were differentially expressed between the atrium and the ventricle in all 6 patients examined. The quotient of the normalized expression from the atrium and ventricle is given, in each case as

Average of all 6 subjects.

The differential expression of the potassium channels TWIK-1, TASK-1, GIRKl, SK2 and PCNl found by means of an array between the atrium and ventricle is verified by quantifying the mRNA in a real-time polymerase chain reaction (10). For this purpose, the total RNA is isolated from the human myocardial samples as described above and 1 μg of each is reacted with 1 unit DNase I (Gibco) for 15 min at room temperature to remove contaminations of genomic DNA. The DNase I is activated by adding 1 μl EDTA (25 inM) and then heating to 65 ° C. (10 min). - The cDNA synthesis is then carried out in the same reaction mixture in accordance with the instructions for the "SUPERSCRIPT-π RT cDNA synthesis kit" (from Gibco) and the reaction volume is diluted with distilled water

Top up 200 μl.

For the PCR, 7.5 μl of the mixture of primer and probe and 12.5 μl of TaqMan reaction solution [Universal Master Mix (from Applied Biosytems]) are added to 5 μl of the diluted cDNA solution, and the final concentration of the primers is 300 nM in each case that of the probe 150 nM. The sequences of the primers and the Geribank accession numbers of the genes analyzed are given in FIG.

Suitable primer and probe sequences were identified with the program Primer Express 5.0 (Applied Biosystems), the PCR is carried out on an ABI Prism SDS-7700 device (Applied Biosystems) according to the manufacturer's instructions. In real-time PCR, the so-called Ct value is recorded, which is obtained for the gene in question in the tissue examined. This corresponds to the cycle in which the fluorescence intensity of the released probe is approx. 10 standard deviations above the background signal. The lower the Ct value, the earlier the reproduction begins, ie the more mRNA is contained in the original sample. To compensate for any fluctuations in the cDNA synthesis, the expression of a so-called “household gene” is also analyzed in all examined tissues. This should be expressed approximately equally strongly in all tissues. For the normalization of the potassium channel expressions, a uniform beta was used for the atrium and ventricles The dCt value is calculated for each gene and each tissue for the graphical representation of the relative m-RNA expression The dCt value is the difference between the Ct value of the examined potassium channel and the Ct value of the household gene In the respective tissue, the relative expression rE is calculated from this value using the following formula: rE = 2 ^{(20 "dct)} This is indicated in FIG. 3 as a dimensionless number.

For the potassium channel TASK-1, the protein expression was analyzed using a commercially available antibody (from Santa Cruz). For this, small pieces of tissue (approx. 50 mg) were homogenized in 1 X PBS (with 1% Triton) and after centrifugation and concentration determination

(BCA-Tet, Pierce) performed a Western blot (10% Nupage gel). The detection was carried out by means of the ECL system (from Amersham) using an HRP-conjugated anti-goat IgG antibody. The exposed film was evaluated densitrometrically in a Bioimager (from Fuji). The result is shown in Fig. 4 as a dimensionless number.

Example 2: Identification of potassium channel modulators

Potassium channel modulators are identified in a cellular assay in which CHO cells recombinantly express the respective ion channel and using the potential-sensitive dye Dye B from the "FLEPR membrane potential assay kit" (Molecular Probes). A depolarization of the cells by a chemical Substance leads to an increased absorption of the dye "Dye B" and thus to an increased intracellular fluorescence intensity. Hyperpolarization of the cell by a chemical substance, on the other hand, leads to a decrease in the dye concentration in the cell and thus also to a decrease in the fluorescence intensity, since the quantum yield of Dye B in aqueous solution is lower. Confluent cells are used for the measurement and, after removal of the medium, are loaded with the dye Dye B in accordance with the instructions of the kit manufacturer (Molecular Probes) at room temperature. The fluorescence measurement is also carried out at room temperature in a fluobox (from Tecan) with an excitation wavelength of 520 nm and an absorption wavelength of 575 nm, as described for example in (11). Example 3; Testing the in vivo effects of potassium channel modulators

The influence of potassium channel modulators on the heart rate is examined in anesthetized rats. For this purpose, male Wistar rats (250-300g) are anesthetized with 10mg / kg thiobutabarbital ip (Inactin, Byk Gulden) and then killed. After the thorax has been opened, the heart is exposed, the right atrium is isolated and stored under a 1g preload in a 30 ° C Krebs-Henseleit solution (in a 10 ml organ bath). This solution is gassed with carbogen (95% 0 ₂ , 5% C0 ₂ ) at pH 7.2-7.4. The atria beat spontaneously and after recording a control period (parameter: frequency), the test substances are applied in a dose series. The change in frequency compared to placebo-treated controls is evaluated per dose.

Example 4: Potassium Channel Modulator Formulations

The potassium channel modulators can be converted in a known manner into the customary formulations, such as tablets, dragées, pills, granules, aerosols, syrups, emulsions, suspensions and solutions, using inert, non-toxic, pharmaceutically suitable excipients or solvents. Here, the therapeutically active compound should in each case be present in a concentration of 0.5 to 90% by weight of the total mixture, i.e. in amounts sufficient to achieve the dosage range indicated.

The formulations are prepared, for example, by stretching the active ingredients with solvents and / or carriers, optionally using emulsifiers and / or dispersants, e.g. if water is used as the diluent, organic solvents can optionally be used as auxiliary solvents.

The application is carried out in the usual way, preferably orally, transdermally, intravenously or parenterally, in particular orally or intravenously. However, it can also be done by inhalation through the mouth or nose, for example with the aid of a spray, or topically via the skin.

In general, it has proven to be advantageous to administer amounts of approximately 0.001 to 10 mg / kg, preferably approximately 0.005 to 3 mg / kg of body weight in the case of oral use in order to achieve effective results.

Nevertheless, it may be necessary to deviate from the quantities mentioned, depending on the body weight or the type of route of administration, on the individual behavior towards the medication, the type of its formulation and the time or

Interval at which the administration takes place. In some cases it may be sufficient to make do with less than the minimum quantity mentioned, while in other cases the specified upper limit must be exceeded. In the case of application of larger quantities, it may be advisable to distribute them in several individual doses over the day.

literature

1. Forth, Henschler, Rummel; General and special pharmacology and toxicology; Urban & Fischer Verlag Munich, 8th edition 2001, 429-433

2. Numaguchi H. et al., Probing the interaction between inactivation gating and Dd-solatol block of HERG, Circ. Res. 11 (2000) 1012-1018.

3. Nattel, S. et al., Evolution, machanisms, and classification of antiarrhyüimic drugs: focus on class iπ actions, Am. J. Cardiol. 84 (1999) 11R-19R.

4. Workmann, A.J. et al., A K (ATP) Channel opener inhibited myocardial reperfusion action potential shortening and arrhythmias.

5. Lesage, F. et al., TWIK-1, a ubiquitous human weakly inward rectifying K ⁺ Channel with a novel structure, EMBO J. 15 (1996) 1004-1011.

6. Duprat, F. et al., TASK, a human background K + Channel to sense external pH variations near physiological pH, EMBO J. 16 (1997) 5464-5471.

7. Stoffel, M. et al., Human G-protein-coupled inwardly rectifying potassium Channel (GIRKl) gene (KCNJ3): localization to chromosome 2 and identification of a simple tandem repeat polymorphism, Genomics 21 (1994) 254-256.

8. Desai, R. et al., Ca ²⁺ -activated K ⁺ channels in human leukemic Jurkat T cells. Molecular cloning, biochemical and functional characterization, J. Biol. Che. 275 (2000) 39954-39963.

9. Tarπkun M. et al., Molecular cloning and characterization of two voltage-gated K + Channel cDNAs from human ventricle, FASEB J. 5 (1991) 331-337.

10. Heid C. et al., Real time quantitative PCR, Genome Res. 6 (1996) 986-9954.

11. EP906572 (B1)

Claims

claims

1. Use of modulators or a modulator of the potassium channels TWIK-1, TASK-1, GIRKl, SK2 or PCNl for the manufacture of a medicament for the treatment and / or prophylaxis of cardiac arrhythmias, arrhythmias, coronary heart diseases or high blood pressure.

2. A method for screening test compounds for the identification of modulators of the potassium channels TWIK-1, TASK-1, GIRKl, SK2 or PCNl, which are suitable for the production of a medicament for the treatment and / or prophylaxis of cardiac arrhythmias (arrhythmias), coronary Heart disease or high blood pressure.

3. A pharmaceutical composition containing one or more modulators of the potassium channels TWTK-1, TASK-1, GIRKl, SK2 or PCNl for the treatment and / or prophylaxis of cardiac arrhythmias, arrhythmias, coronary heart diseases or high blood pressure.

4. Modulator of the potassium channels TWIK-1, TASK-1, GIRKl, SK2 or PCNl for the treatment and / or prophylaxis of arrhythmias (arrhythmias), coronary heart diseases or high blood pressure.

5. Use of modulators of gene products, which are differentially expressed in the human heart between the left atrium and left ventricle, for the production of a medicament for the treatment of arrhythmias (arrhythmias), coronary heart diseases, or high blood pressure.