Method and Kit for Assessment of Sodium channel-Related Anti- or Pro-Arrhythmic Potential of compounds

Abstract

The present invention relates to an in vitro method for evaluating the anti- or pro-arrhythmic potential, cardiotoxicity and/or modulation capacity of cardiomyocyte function of compound(s). The present invention also relates to compound(s) identified or evaluated in the method of the invention for use in the treatment of a heart disease. The present invention further relates to the use of the density change of cardiac Nav 1.5 sodium channels in intercalated discs of cardiomyocytes as marker and/or diagnostic for the anti- or pro-arrhythmic potential of a compound, the cardiotoxicity of a compound or modulation capacity of cardiomyocyte function by said compound, and/or in preclinical assessment for cardiac liability of compounds and cardio-safety assessment. The present invention further relates to a kit for evaluating the anti- or pro-arrhythmic potential, cardiotoxicity and/or modulation capacity of cardiomyocyte function of compound(s).

Claims

1. An in vitro method for evaluating the anti- or pro-arrhythmic potential, cardiotoxicity and/or modulation capacity of cardiomyocyte function of compound(s), comprising the steps of: (a) providing cardiomyocytes, (b) incubating/culturing the cardiomyocytes of step (a); (c) providing a compound to be tested; (d) adding the compound of step (c) to the cardiomyocytes of step (b), and incubating the compound with the cardiomyocytes, (e) detecting the intercalated discs of cardiomyocytes, and thereby determining the area of the intercalated discs in the cardiomyocyte(s), (f) determining the density of the cardiac sodium channels Nav1.5 within said area of the intercalated discs, and (g) determining whether there is a density change of the cardiac sodium channels Nav1.5 in the area of the intercalated discs as determined in step (e), by comparing the density of the cardiac sodium channels Nav1.5 determined in step (f) with the density determined in isolated cardiomyocytes where in step (d) no compound was added, wherein a relative density change indicates that the tested compound has an anti- or pro-arrhythmic potential, cardiotoxicity and/or modulation capacity of cardiomyocyte function.

2. The method of claim 1, wherein the cardiomyocytes are selected from adult cardiomyocytes, primary cardiomyocytes, embryonic stem (ES) cell-derived cardiomyocytes, induced pluripotent stem (iPS) cell-derived cardiomyocytes, human adult cardiomyocytes, human progenitor cell-derived cardiomyocytes, human embryonic stem (hES) cell-derived cardiomyocytes, and human induced pluripotent stem (hiPS) cell-derived cardiomyocytes.

3. The method of claim 1, wherein in step (d) conditions and/or parameters for adding the test compound are varied, such that the test compound is added in different concentrations, in different solvent, in different culture media, at different incubation temperatures, at different times after cell preparation, and/or in different intervals in pulse-chase analyses.

4. The method of claim 1, wherein the detection of the intercalated discs of cardiomyocytes in step (e) is carried out by binding of at least one detection compound to one or several components of the adherens junction, desmosome junction, and/or intercalated disc associated protein-complex.

5. The method of claim 4, wherein the at least one detection compound used in step (e) to detect the intercalated discs of cardiomyocytes is selected from: labeled antibodies, labeled nucleic acid aptameres, labeled protein aptameres, labeled protein binding substances, labeled viral proteins, and tagged gene expression products of vectors.

6. The method of claim 1, wherein in step (f) the density of the cardiac sodium channels Nav1.5 is determined using at least one of the following detection compounds: labeled antibodies, labeled nucleic acid aptameres, labeled protein aptameres, labeled protein binding substances and tagged gene expression products of vectors.

7. The method of claim 5, wherein the label(s) of the detection compounds are selected from: fluorophores, radionuclides, luminescent dyes and enzymes.

8. The method of claim 1, comprising imaging/detection via microscopy, photostimulated luminescence, and radionuclide imaging.

9. The method of claim 1, wherein determining the density of the cardiac sodium channels Nav1.5 within the area of the intercalated discs in step (f) comprises: (1) detecting the area containing cardiac sodium channels Nav1.5 in the cardiomyocyte(s), and/or (2) determining the area of the intercalated discs which contain Nav1.5 channels.

10. A method for assessing anti- or pro-arrhythmic potential of a compound, assessing cardiotoxicity of a compound or modulation capacity of cardiomyocyte function, and/or preclinical assessment for cardiac liability of compounds/cardio-safety assessment; wherein said method utilizes the density change of cardiac sodium channels in intercalated discs of cardiomyocytes.

11. The method of claim 10, wherein the density change of the sodium channels Nav1.5 in the intercalated discs of cardiomyocytes is determined.

12. The method of claim 10, comprising the quantitative in vitro determination of the density change of the sodium channels Nav1.5 in the intercalated discs of cardiomyocytes after interaction with a compound by comparing the density of the sodium channels Nav1.5 in the intercalated discs of treated cardiomyocytes with the density of the sodium channels Nav1.5 in the intercalated discs of untreated cardiomyocytes.

13. The method of claim 10, wherein the cardiomyocytes are selected from adult cardiomyocytes, primary cardiomyocytes, embryonic stem (ES) cell-derived cardiomyocytes, induced pluripotent stem (iPS) cell-derived cardiomyocytes, human adult cardiomyocytes, human progenitor cell-derived cardiomyocytes, human embryonic stem (hES) cell-derived cardiomyocytes, and human induced pluripotent stem (hiPS) cell-derived cardiomyocytes.

14. A kit for evaluating the anti- or pro-arrhythmic potential, cardiotoxicity and/or modulation capacity of cardiomyocyte function of compound(s), comprising (i) means for detecting the intercalated discs of cardiomyocytes and determining the area of intercalated discs in the cardiomyocyte(s), (ii) means for determining the density of the cardiac sodium channels Nav1.5, (iii) means and/or instructions for determining the density change of the cardiac sodium channels Nav1.5 within said area of the intercalated discs, and (iv) optionally, means and/or instruction for obtaining suitable cardiomyocytes.

15. A method for the treatment of heart disease wherein said method comprises administering to a subject in need of such treatment a compound identified or evaluated in a method of claim 1 as having an anti- or pro-arrhythmic potential, cardiotoxicity and/or modulation capacity of cardiomyocyte function.

16. The method, according to claim 2, wherein said adult cardiomyocytes and primary cardiomyocytes are isolated from cardiac tissue of a vertebrate; and the human adult cardiomyocyte is derived from a human biopsy or explanted human heart tissue.

17. The method, according to claim 4, wherein the adherens junction is selected from N-cadherin, plakoglobin, plakophilin, CAR, LIMP-2, vinculin, metavinculin, ZO-1, mXinα, α-actinin, α-E-catenin, α-T-catenin, and β-catenin; the desmosome junction is selected from desmocollin, desmoglein, plakoglobin, β-catenin, plakophilin, desmoplakin, desmin, and myozap; and the intercolated disc associated protein-complex is selected from ankyrin G, synapse-associated protein 97 (SAP97), syntrophin/dystrophin, Ca.sup.2+/calmodulin-dependent protein kinase II (CAMKII), and connexin 43.

18. The kit, according to claim 14, wherein the means (i) for detecting the intercalated discs of cardiomyocytes is selected from a labeled anti-cadherin antibody, or a primary anti-cadherin antibody and a secondary labeled antibody, the means (ii) for determining the density of the cardiac sodium channels Nav1.5 is selected from a labeled anti-Nav1.5 antibody, or a primary anti-Nav1.5 antibody and a secondary labeled antibody.

19. The method, according to claim 15, used to treat a cardiac arrhythmia, cardiomyopathy, failing heart and/or cardiac hypertrophy.

20. The method, according to claim 15, wherein the compound is flecainide.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0187] FIG. 1 schematically shows the steps of a preferred embodiment of the method according to the invention (A) for cultivating the cardiomyocytes in vitro, incubation with the compound to be tested, immunocytochemical staining with antibodies and confocal microscopy. Shown are the confocal picture of the Nav1.5 sodium channels (B) and panCadherin staining of the intercalated discs (C) as well as their merge (D).

[0188] FIGS. 2 and 3 show how the confocal pictures are evaluated.

[0189] The panCadherin staining (FIG. 2A) is used for determining the Regions of Interest (ROI) (FIG. 2B; red areas). The relative density of the Nav1.5 stainings at 20% (FIG. 2C), 50% (FIG. 2D) and 80% (FIG. 2E) laser intensity were determined in the ROI (F,G,H; yellow areas).

[0190] The D.sub.20, D.sub.50 and D.sub.80 values of the relative Nav1.5 density are plotted in a xy-coordinate system (FIG. 3) for determining their intensity-dependent linear gradient.

EXAMPLES

Example 1

1. Material & Methods

1.1 Mouse Adult Cardiomyocyte Isolation

[0191] Cardiomyocytes isolation was performed largely according to Liao & Jain (2007). Briefly, the mice were injected with 200 IU heparin i.p. prior to sacrifice. The thoracic chamber was opened and a cannula with perfusion solution was inserted from the atria. The heart was harvested and perfused in the Langendorff system with Perfusion Buffer at 37° C. for 5 min, followed by Digestion Buffer until the heart muscle was pale and some signs of extracellular matrix dissociation appeared. The heart was perfused for additional 5 min with Perfusion Buffer to stop the dissociation. The cardiomyocytes were mechanically dissociated in TB-A and then plated on ECM-coated (Sigma cat. no. E-1270) petri dish (Zell-Kontakt cat. no. 5160-30). The extracellular calcium concentration was increased gradually in three consecutive steps from 0 mM (in TB-A) to 0.06 mM, 0.24 mM and 1.2 mM every 5 min. The cells were washed once with TB-B, transferred to round coverslips (on a 24-well cell culture plate; TPP cat. no. 92024) in Plating Medium. After 1 h incubation in a cell incubator at 37° C. at 5% CO.sub.2 the medium was changed to Culture Medium and all further experiments were perforated in Culture Medium under the same conditions.

Solutions (Indications in mM):

[0192] Perfusion Buffer: 135 NaCl, 4 KCl, 1 MgCl.sub.2, 10 HEPES, 0.33 NaH.sub.2PO.sub.4, 10 glucose, 20 2,3-butanediones monoxime (Sigma cat. no. B0753), 5 Taurine, pH7.2 at 37° C. [0193] Digestion Buffer: 0.3 mg/g body weight collagenase D (Roche cat. no. 11088858001), 0.4 mg/g body weight collagenase B (Roche cat. no. 11088807001) and 0.05 mg/g body weight protease XIV (Sigma cat. no. P5147) in 25 ml Perfusion Buffer. [0194] Transfer Buffer A (TB-A): 135 NaCl, 4 KCl, 1 MgCl.sub.2, 10 HEPES, 0.33 NaH.sub.2PO.sub.4, 5.5 glucose, 10 2,3-butanediones monoxime (Sigma cat. no. B0753), 5 mg/ml bovine serum albumin (Sigma cat. no. A6003), pH7.4 at 37° C. [0195] Transfer Buffer B (TB-B): 137 NaCl, 5.4 KCl, 1.8 CaCl.sub.2, 0.5 MgCl.sub.2, 10 HEPES, 5.5 glucose, pH 7.4 at 37° C. [0196] Plating Medium: Minimal Essential Medium, 100 U/ml penicillin-streptomycin, 2 L-glutamine, 10 2,3-butanedione monoxime (Sigma cat. no. B0753), 5% fetal calf serum. [0197] Culture Medium: Minimal Essential Medium, 100 U/ml penicillin-streptomycin, 2 L-glutamine, 0.1 mg/ml bovine albumin (Sigma cat. no. A6003).

1.2 Application of Chemical Compounds to Cardiomyocytes in Culture

[0198] Chemical or pharmaceutical compounds, e.g. fleicainide (Sigma Aldrich cat. no. F0120000), ajmaline (MP Biomedicals cat. no. 05212414), were dissolved in Culture Medium at 5 different concentrations, such as 0.01 mM, 0.1 mM, 1 mM, 10 mM, 100 mM, and applied to cardiomyocytes in 5 different wells, respectively. At the same time the medium in a control well was replaced with fresh Culture Medium. The cells were further incubated for up to 8 h in a cell incubator at 37° C. at 5% CO.sub.2.

1.3 Immuncytochemistry

[0199] For immuncytochemical detection of intercalated discs and Nav1.5 cardiac sodium channels cardiomyocytes were fixed in 0.5% TritonX/PBS for 10 min, followed by incubation in 0.1M glycin/PBS for 1 h. Cardiornyocytes were blocked with 2% BSA/PBS (bovine serum albumin Sigma cat. no. A6003) for 1 h and incubated with a solution composed of 1:50 anti-Nav1.5 (Alomone Labs cat. no. ASC-005) and 1:500 anti-panCadherin (Abeam cat. no. ab22744) in 2% BSA/PBS overnight. The next day cells were washed with PBS for 15 min and incubated with a 1:500 dilution of labeled secondary antibodies MFP-A1034 and MFP-A2424 (MoBiTec) in 2% BSA/PBS for 4 h at room temperature. The cells were washed with PBS for 15 min, the coverslips with cells were mounted on a microscope slide in AF1 antifadent mountant solution (Citifluor).

1.4 Confocal Microscopy and Image Processing

[0200] Confocal images of red and green immunofluorescence of stained cardiomyocytes were acquired, e.g. on a Leica confocal laser scanning unit TCS NT, which is coupled to a Leica DM IRB microscope. Acquisition of image series was performed, e.g. using TCS NT (Leica, Hedelberg, Germany) software. Fluorescence signals of Nav1.5 staining were recorded at 20%, 50% and 80% laser intensity, and fluorescence signals of cadherin was recorded at 80% laser intensity to generate the Region of Interest (ROI) (modified from Yampolsky et al., 2010, a). All images were digitally saved and later processed with Image) (NIH, Bethesda, Md., USA) software using proprietary quantification methods and standard Image) plug-ins.

1.5 Image Quantification and Nav1.5 Density Change Assessment

[0201] ROI were set as defined by the fluorescence signals of the cadherin staining, which represented the intercalated discs. The areas containing the fluorescence signal of Nav1.5 staining were measured separately in images recorded at 20%, 50% and 80% laser intensities, while applying the same threshold value (X) for all images (modified from Yampolsky et al., 2010, b). Nav1.5 density values D.sub.20, D.sub.50 and D.sub.80 were calculated by division of respective area values containing Nav1.5 by the ROI area values as determined by measurement of the cadherin signals:

[00004] $D_{20} .Math. : .Math. .Math. relative .Math. .Math. Nav .Math. .Math. 1.5 .Math. .Math. density .Math. .Math. at .Math. .Math. 20 .Math. % .Math. .Math. laser .Math. .Math. intensity = \frac{area .Math. .Math. of .Math. .Math. Nav .Math. .Math. 1.5 .Math. .Math. signal .Math. .Math. at .Math. .Math. threshold .Math. .Math. X}{R .Math. .Math. O .Math. .Math. I .Math. .Math. area}$ $D_{50} .Math. : .Math. .Math. relative .Math. .Math. Nav .Math. .Math. 1.5 .Math. .Math. density .Math. .Math. at .Math. .Math. 50 .Math. % .Math. .Math. laser .Math. .Math. intensity = \frac{area .Math. .Math. of .Math. .Math. Nav .Math. .Math. 1.5 .Math. .Math. signal .Math. .Math. at .Math. .Math. threshold .Math. .Math. X}{R .Math. .Math. O .Math. .Math. I .Math. .Math. area}$ $D_{80} .Math. : .Math. .Math. relative .Math. .Math. Nav .Math. .Math. 1.5 .Math. .Math. density .Math. .Math. at .Math. .Math. 80 .Math. % .Math. .Math. laser .Math. .Math. intensity = \frac{area .Math. .Math. of .Math. .Math. Nav .Math. .Math. 1.5 .Math. .Math. signal .Math. .Math. at .Math. .Math. threshold .Math. .Math. X}{R .Math. .Math. O .Math. .Math. I .Math. .Math. area}$

[0202] The intensity-dependent linear slope of Nav1.5 fluorescence signal values was calculated for cardiomyocytes incubated with the test compounds as follows:

[00005] $slope .Math. .Math. of .Math. .Math. Nav .Math. .Math. 1.5 .Math. .Math. fluorescence .Math. .Math. signal .Math. .Math. values = [\frac{(D_{80} - D_{50})}{80 .Math. % - 50 .Math. %} + \frac{(D_{50} - D_{20})}{50 .Math. % - 20 .Math. %} + \frac{(D_{80} - D_{20})}{80 .Math. % - 20 .Math. %}] : .Math. 3$

[0203] The slope values calculated for cardiomyocytes treated with different concentrations of the test compounds were divided by the slope values for untreated cardiomyocytes:

[00006] $Nav .Math. .Math. 1.5 .Math. .Math. density .Math. .Math. change .Math. .Math. in .Math. .Math. % = \frac{slope .Math. .Math. of .Math. .Math. Nav .Math. .Math. 1.5 .Math. .Math. fluorescence .Math. .Math. signal .Math. .Math. values, treated}{slope .Math. .Math. of .Math. .Math. Nav .Math. .Math. 1.5 .Math. .Math. fluourescence .Math. .Math. signal .Math. .Math. values, untreated} \times 100$

[0204] The comparison of slope values provides information on the degree of change in Nav1.5 density as a result of interaction with the test compounds at different concentrations and indicates an impact of the compound on cardiac conduction.

[0205] The features disclosed in the foregoing description, in the claims and/or in the accompanying drawings may, both separately and in any combination thereof, be material for realizing the invention in diverse forms thereof

REFERENCES

[0206] Brown A M. HERG block, QT liability and sudden cardiac death. Novartis Found Symp. 2005; 266:118-31; discussion 131-5, 155-8. Review. [0207] Cavero I, Crumb W. ICH S7B draft guideline on the non-clinical strategy for testing delayed cardiac repolarisation risk of drugs: a critical analysis. Expert Opin Drug Saf. 2005 May; 4(3):509-30. [0208] CAS Trial II Investigators. Effect of the antiarrhythmic agent moricizine on survival after myocardial infarction. N Engl J Med, 327(4), 227-33, (1992). [0209] Echt D S, Liebson P R, Mitchell L B, Peters R W, Obias-Manno D, Barker A H, . . . & Richardson D W. Mortality and morbidity in patients receiving encainide, flecainide, or placebo: the Cardiac Arrhythmia Suppression Trial. New England Journal of Medicine, 324(12), 781-788, (1991). [0210] Haverkamp W, Eckardt L, Kirchhof P, Mönnig G, Schulze-Bahr E, Wedekind H, Borggrefe M, Madeja M, Breithardt G. New knowledge in arrhythmogenesis—role of ion channels and genetic aspects. Z Kardiol. 2000; 89 Suppl 10:2-9; discussion 9-10. Review. [0211] Liao, R., Jain, M. Isolation, culture, and functional analysis of adult mouse cardiomyocytes. Methods in Molecular Medicine. 139:251-62 (2007). [0212] Maguy A, Hebert T E, Nattel S. Involvement of lipid rafts and caveolae in cardiac ion channel function. Cardiovasc Res. 2006 Mar. 1; 69(4):798-807. [0213] Watanabe H, Yang T, Stroud D M, Lowe J S, Harris L, Atack T C, Wang D W, Hipkens S B, Leake B, Hall L, Kupershmidt S, Chopra N, Magnuson M A, Tanabe N, Knollmann B C, George A L Jr, Roden D M. Striking In vivo phenotype of a disease-associated human SCNSA mutation producing minimal changes in vitro. Circulation. 2011 Aug. 30; 124(9):1001-11. [0214] Yampolsky, P., Pacifici, P. G., Witzemann, V. Differential muscle-driven synaptic remodeling in the neuromuscular junction after denervation. European Journal of Neuroscience, 31(4), 646-658, (2010, a). [0215] Yampolsky, P., Pacifici, P. G., Lomb, L., Giese, G., Rudolf, R., Roder, I. V., Witzemann, V. Time lapse in vivo visualization of developmental stabilization of synaptic receptors at neuromuscular junctions. Journal of Biological Chemistry, 285(45), 34589-34596, (2010, b). [0216] Xi B, Wang T, Li N, Ouyang W, Zhang W, Wu J, Xu X, Wang X, Abassi Y A. Functional cardiotoxicity profiling and screening using the xCELLigence RTCA Cardio System. J Lab Autom. 2011 December; 16(6):415-21.

Method and Kit for Assessment of Sodium channel-Related Anti- or Pro-Arrhythmic Potential of compounds

Inventors

Cpc classification

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A61K31/4458

HUMAN NECESSITIES

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G01N33/6872

PHYSICS

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G01N33/5061

PHYSICS

Classification Explorer

G01N33/5073

PHYSICS

International classification

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G01N33/50

PHYSICS

Classification Explorer

A61K31/4458

HUMAN NECESSITIES

Abstract

Claims

Description