Abstract
A means for treating cardiovascular disease, in particular chronic heart failure, especially in terms of a more specific treatment preventing or minimizing pathological hypertrophic signaling while leaving cardiac contractility largely intact. For this purpose the present invention provides a means for treating cardiovascular disease, in particular chronic heart failure, the means specifically inhibiting or causing inhibition of components of the β1-AR/cAMP pathway generating cAMP resulting from activation of the β1-adrenoceptor on the cardiomyocyte cell crest.
Claims
1. A β1-AR/cAMP microdomain based therapeutic for preventing or minimizing pathological hypertrophic signalling while leaving cardiac contractility largely intact, comprising a) a carrier carrying immobilized on its surface a binding molecule specifically blocking the β1-adrenoceptor, the binding molecule being selected from beta-blockers, antibodies and aptamers, the carrier having a size excluding it from entering T-tubules, or b) a complex of a binding molecule, the binding molecule being selected from antibodies specifically blocking the β1-adrenoceptor, the complex having a size excluding it from entering T-tubules.
2. The therapeutic according to claim 1, wherein the binding molecule is a beta blocker specifically blocking the β.sub.1-adrenoceptor on the cardiomyocyte cell crest.
3. The therapeutic according to claim 1, wherein the binding molecule is a binding molecule specifically binding to and blocking the β.sub.1-adrenoceptor on the cardiomyocyte cell crest.
4. The therapeutic according to claim 3, wherein the binding molecule is an antibody or an aptamer specifically binding to and blocking the β.sub.1-adrenoceptor on the cardiomyocyte cell crest.
5. The therapeutic s according to claim 1, wherein the therapeutic is a nanoparticle having a diameter of more than 11 nm and carrying immobilized on its surface a binding molecule specifically blocking the β.sub.1-adrenoceptor.
6. The therapeutic according to claim 1, the binding molecule being a genetically encoded inhibitor of the β.sub.1-AR/cAMP pathway engineered to localize at the cell crest.
7. The therapeutic according to claim 1, the binding molecule being a pH dependent beta-blocker specifically inhibiting the β.sub.1-adrenoceptor on the cardiomyocyte cell crest.
8. A medicament comprising a therapeutic according to claim 1.
9. A β1-AR/cAMP microdomain based method of therapeutic treatment for preventing or minimizing pathological hypertrophic signalling while leaving cardiac contractility largely intact, comprising administering to a patient in need of such treatment a therapeutically effective amount of a) a carrier carrying immobilized on its surface a binding molecule specifically blocking the β1-adrenoceptor, the binding molecule being selected from beta-blockers, antibodies and aptamers, the carrier having a size excluding it from entering T-tubules, or b) a complex of a binding molecule, the binding molecule being selected from antibodies specifically blocking the β1-adrenoceptor, the complex having a size excluding it from entering T-tubules.
10. The method of therapeutic treatment according to claim 9, wherein the cardiovascular disease is chronic heart failure.
11. The method of therapeutic treatment according to claim 9, wherein the cardiovascular disease is coronary artery disease or cardiac arrhythmias.
12. The therapeutic according to claim 4, wherein the antibody is a monoclonal antibody.
13. The therapeutic according to claim 1, the therapeutic being or comprising a carrier carrying covalently bound on its surface a binding molecule specifically blocking the β.sub.1-adrenoceptor, the carrier having a size excluding it from entering T-tubules.
14. The therapeutic according to claim 1, wherein the therapeutic is a nanoparticle having a diameter of more than 11 nm and carrying covalently bound on its surface a binding molecule specifically blocking the β.sub.1-adrenoceptor.
Description
(1) In the following, the invention is described for illustration purposes only in more detail.
(2) FIG. 1 Simplified and schematic cross-sectional view of a part of a cardiomyocyte (CM) showing T-tubules and crest regions of the sarcolemma with crest β.sub.1-ARs (large closed circles) and T-tubular β.sub.1-ARs (large open circles) and part of the SR (sarcoplasmic reticulum) with Protein kinase A type II (PKA II) and phosphodiesterase type 4 (PDE4).
(3) FIG. 2 Principle of the scanning ion conductance microscopy (SICM) combined with a FRET biosensor for cAMP (A). This technique uses a ligand containing nanopipette (size ˜50 nm) for localization and precise local stimulation of β-AR populations at different CM membrane structures such as T-tubuli and cell crests. B. Receptor-triggered cAMP signals are detected intracellularly by a cytosolic FRET based biosensor. C. This technique showed that β.sub.1-AR is located in both T-tubuli and crests and that both receptor pools activate cytosolic cAMP synthesis to a similar degree. ISO, β-AR agonist isoproterenol. ICI, β.sub.2-AR blocker ICI 118,551 locally applied via SICM pipette to marked locations (white arrows).
(4) FIG. 3 Two distinct β.sub.1-AR populations revealed by SICM/FRET. A-C. Rat ventricular CMs expressing nuclear localized Epac1-NLS sensor show stronger β.sub.1-AR/cAMP responses stimulated by the crest than T-tubular pool of receptors. D-F. The same cells expressing a nuclear PKA activity reporter AKAR3-NLS show much stronger nuclear PKA activity induced by β.sub.1-AR located in the crest, as compared to receptors stimulated in the T-tubules.
(5) FIG. 4 Cardiomyocyte contractility responses measured as an amplitude of calcium transient upon application of β.sub.1-AR stimulus (ISO+ICI) to T-tubuli or cell crests. Calcium transients are much stronger stimulated by T-tubular β.sub.1-AR suggesting that this receptor pool is more strongly involved in the regulation of cellular contractility.
(6) FIG. 5. Measurements of nuclear PKA activity in failing ventricular cardiomyocytes isolated from rats with a chronic heart failure model 16 weeks after myocardial infarction (A) and from patients with hypertrophic cardiomyopathy undergoing aortic valve replacement surgery. SICM/FRET measurements were performed as described in FIG. 3D-F.
(7) FIG. 6. Pharmacological characterization of atenolol coated nanoparticles (NP). A. Schematic representation of an atenolol coated gold nanoparticle. B-C. Experimental data with HEK293 cells stably expressing β.sub.1-AR and the cAMP biosensor Epac1-camps. Using FRET-based cAMP assay it could be shown that atenolol coated (Atenolol NP) but not bare control (Contol NP) effectively inhibit ISO-induced cAMP increase.
(8) FIG. 7. Experimental data for cardiomyocyte hypertrophy (A) and cell contractility (B) assays performed with freshly isolated adult rat ventricular cardiomyocytes (CM) using atenolol coated nanoparticles (Atenolol NP) and control nanoparticles. Atenolol NP can block cardiomyocyte hypertrophy but do not inhibit contractility.
(9) FIG. 1 shows a simplified and schematic cross-sectional view of a part of a cardiomyocyte (CM). FIG. 1 shows T-tubules 1 being invaginations of the sarcolemma 8 and crest regions 2 of the sarcolemma 8 with β.sub.1-ARs 3 (large closed circles) and T-tubular β.sub.1-ARs 4 (large open circles) and part of the SR 5 (sarcoplasmic reticulum) with Protein kinase A type II 6 (PKA II) and phosphodiesterase type 4 7 (PDE4).
(10) To study functional β-AR localization, a combination of scanning ion conductance microscopy (SICM) with FRET based recording of intracellular cAMP called SICM/FRET was used (Nikolaev, V. O., Moshkov, A., Lyon, A. R., Miragoli, M., Novak, P., Paur, H., Lohse, M. J., Korchev, Y. E., Harding, S. E., & Gorelik, J. 2010. Beta2-adrenergic receptor redistribution in heart failure changes cAMP compartmentation. Science, 327, 1653-1657) (FIG. 2). This method allows imaging of live CM membrane structures and associated signalling with nanometre precision. Real time dynamics of cAMP in intact cardiomyocytes (CMs) can be studied using highly sensitive biosensors based on Förster Resonance Energy Transfer (FRET). Such sensors contain a single cyclic nucleotide binding domain fused to a pair of fluorescent proteins and change their conformation upon cAMP binding (Nikolaev, V. O., Bunemann, M., Hein, L., Hannawacker, A., & Lohse, M. J. 2004. Novel single chain cAMP sensors for receptor-induced signal propagation. J Biol Chem, 279, 37215-37218), resulting in a decrease of FRET (FIG. 2B). They can be expressed in transgenic animals in CM-specific (Nikolaev, V. O., Bünemann, M., Schmitteckert, E., Lohse, M. J., & Engelhardt, S. 2006. Cyclic AMP imaging in adult cardiac myocytes reveals far-reaching beta1-adrenergic but locally confined beta2-adrenergic receptor-mediated signaling. Circ Res, 99, 1084-1091) and ubiquitous manner (Calebiro, D., Nikolaev, V. O., Gagliani, M. C., de Filippis, T., Dees, C., Tacchetti, C., Persani, L., & Lohse, M. J. 2009. Persistent cAMP-signals triggered by internalized G-protein-coupled receptors. PLoS Biol, 7, e1000172.) to allow live cell cAMP imaging with unprecedented spatio-temporal resolution. Interestingly, when using such cytosolic cAMP biosensor in SICM/FRET experiments upon local activation of β.sub.1-ARs in T-tubules or on cell crests, no difference in cytosolic β.sub.1AR/cAMP responses between these two receptor pools could be observed (FIG. 2C). Recently, several localized versions of cAMP biosensors targeted to the proximity of major Ca-handling proteins such as LTCC, RyR and SERCA (Perera, R. K., Sprenger, J. U., Steinbrecher, J. H., Hubscher, D., Lehnart, S. E., Abesser, M., Schuh, K., El-Armouche, A., & Nikolaev, V. O. 2015. Microdomain switch of cGMP-regulated phosphodiesterases leads to ANP-induced augmentation of beta-adrenoceptor-stimulated contractility in early cardiac hypertrophy. Circ Res, 116, 1304-1311; Sprenger, J. U., Perera, R. K., Steinbrecher, J. H., Lehnart, S. E., Maier, L. S., Hasenfuss, G., & Nikolaev, V. O. 2015. In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease. Nat Commun, 6, 6965) and to the nucleus have been developed. This allowed microdomain-specific imaging of real time cAMP dynamics. Using these microscopy tools combined with an in vivo mouse model of early HF, it could be uncovered that the disease leads to redistribution of several PDEs between different membrane domains associated with T-tubuli which affects β.sub.1-AR stimulated cardiac contractility (Perera, R. K., Sprenger, J. U., Steinbrecher, J. H., Hubscher, D., Lehnart, S. E., Abesser, M., Schuh, K., El-Armouche, A., & Nikolaev, V. O. 2015. Microdomain switch of cGMP-regulated phosphodiesterases leads to ANP-induced augmentation of beta-adrenoceptor-stimulated contractility in early cardiac hypertrophy. Circ Res, 116, 1304-1311). Early HF also leads to an impairment of the direct β.sub.1-AR—SERCA microdomain communication (Sprenger, J. U., Perera, R. K., Steinbrecher, J. H., Lehnart, S. E., Maier, L. S., Hasenfuss, G., & Nikolaev, V. O. 2015. In vivo model with targeted cAMP biosensor reveals changes in receptor-microdomain communication in cardiac disease. Nat Commun, 6, 6965), further contributing to the contractile dysfunction.
(11) To test the hypothesis, that there are at least two distinct populations of β.sub.1-ARs, one located on the crest and stronger stimulating hypertrophy-associated pools of cAMP, and another one found in T-tubules which is predominantly associated with the regulation of CM contractility, a nuclear targeted cAMP sensor Epac1-NLS and a nuclear targeted PKA activity reporter AKAR3-NLS (Haj Slimane, Z., Bedioune, I., Lechene, P., Vain, A., Lefebvre, F., Mateo, P., Domergue-Dupont, V., Dewenter, M., Richter, W., Conti, M., El-Armouche, A., Zhang, J., Fischmeister, R., & Vandecasteele, G. 2014. Control of cytoplasmic and nuclear protein kinase A by phosphodiesterases and phosphatases in cardiac myocytes. Cardiovasc Res, 102, 97-106) were used in SICM/FRET experiments under local β.sub.1-AR stimulation in either cardiomyocyte crests or T-tubules. Using these sensors, it could clearly be shown that β.sub.1-ARs stimulated at the crest increased cAMP (FIGS. 3A-C) and PKA activity (FIG. 3D-F) in the nucleus much stronger than the receptors stimulated in T-tubules, suggesting that β.sub.1-AR pool located on the crest is directly associated with the pathological cAMP signalling. In contrast, stimulation of β.sub.1-ARs localized in T-tubules led to much stronger contractile responses than those measured after receptor stimulation on cell crests (FIG. 4).
(12) In order to be applicable in terms of possible medication, the above mentioned mechanism involving two β.sub.1-AR receptor pools should be active not only in healthy but also in failing myocytes. FIG. 5 shows SICM/FRET experiments performed as described in FIG. 3D-F with failing rat (FIG. 5A) and failing human (FIG. 5B) myocytes. In both cases, a similar clear difference between receptors stimulated on the crests and in T-tubuli can still be observed. This suggests that the disease, apart from some desensitization (reduction of signal amplitude) does not change the therapeutically interesting pi-AR/cAMP pools targeted by the means of this invention.
(13) FIGS. 6 and 7 show experimental data with beta-blocker coated nanoparticles.
(14) For the preparation of beta-blocker coated nanoparticles 18 nm gold nanoparticles were coated using an amphiphilic polymer poly(isobutylene-alt-maleic anhydride) which was covalently linked to atenolol. Alkylamine chains of the polymer backbone were chemically linked by the direct amidation between maleic anhydride and the amino-ligand dodecylamine which exhibits hydrophobic interaction with gold nanoparticle (Lin, C. A., Sperling, R. A.; Li, J. K.; Yang, T. Y.; Li, P. Y.; Zanella, M.; Chang, W. H.; Parak, W. J., Design of an amphiphilic polymer for nanoparticle coating and functionalization. Small 2008, 4, 334-341). In the second reaction step, atenolol was covalently bound to the polymer via aminogroup and direct amidation (FIG. 6A). The chemicals were typically used at the ratio 2:1:0.5 or 2:1:2 of maleic anhydride/dodecylamine/atenolol. After the synthesis, coated particles were washed, characterized for their size and stability, while last washthrough steps were tested for the absence of atenolol.
(15) FIG. 6B shows the pharmacological characterization of nanoparticles in HEK293 cells stably expressing β.sub.1-ARs using a FRET-based cAMP assay. Addition of the beta-adrenergic agonist isoproterenol (ISO, 0.1 nM) leads to an increase of CFP/YFP ratio indicating an increase in intracellular cAMP. This increase is blocked by pretreatment (20 min) with 30 nM of atenolol coated nanoparticles, while uncoated control 18 nm gold nanoparticles have no effect. Data analysis for these experiments is presented in FIG. 6C. Atenolol coated nanoparticles are almost as efficacious in blocking β.sub.1-AR dependent cAMP synthesis as the pure beta-blocker atenolol (used at 30 nM).
(16) Further experiments with adult rat ventricular CMs were performed in order to evaluate the effect of beta-blocker coated nanoparticles on hypertrophy and cell contractility (see FIG. 7). Freshly isolated adult rat ventricular CMs were cultured for one day and preincubated for 1 hour with bare gold 18 nm nanoparticles (Control NP) or 18 nm gold nanoparticles coated with covalently bound beta-blocker atenolol (Atenolol NP) both at 30 nM. To induce hypertrophy, CMs were then concomitantly stimulated for 24 h with 10 nM isoproterenol (ISO). Cell surface areas were then measured for 80 cells per group. The experiments showed that atenolol coated nanoparticles prevent cardiomyocyte hypertrophy (FIG. 7A).
(17) Freshly isolated adult rat ventricular CMs were incubated for 20 min with atenolol (100 nM), atenolol coated or control nanoparticles (both at 30 nM) and cell contractility was measured using edge detection method (IonOptix) at 1 Hz pacing frequency with and without stimulation with 10 nM isoproterenol. The experiments showed that atenolol coated nanoparticles in contrast to pure atenolol do not decrease cell contractility (FIG. 7B).
