Peptides for treating cardiac diseases

Abstract

The present invention relates to peptides with 11-12 amino acids for use thereof as a medicine in cardiac diseases, especially ventricular arrhythmias in patients with cardiac hypertrophy, cardiac insufficiency (CI) and / or hereditary loss-of-function syndromes in the Nav1.5 or Kir2.1 channels. The peptide with 11 amino acids and the synthetic analogues with 11-12 amino acids include a nuclear localisation signal, activate KCNJ2 transcription, prevent the decrease in the density of INa and IK1 caused by CI, and significantly increase the density of INa and IK1 in cases of CI. The invention also includes cDNA molecules that encode these peptides, the vectors and / or the cells containing them and pharmaceutical compositions containing same for use thereof in the prevention and / or treatment of cardiac diseases.

Description

[0001]

[0002] Peptides for the treatment of heart disease

[0003] TECHNICAL SECTOR

[0004] The present invention falls within the field of biotechnology and peptide synthesis with application in the pharmaceutical sector. More specifically, it relates to peptides with properties that modulate ionic currents in cardiomyocytes.

[0005] BACKGROUND OF THE INVENTION

[0006] Heart failure (HF) is a highly prevalent syndrome characterized by high morbidity and mortality (approximately 50% in the first 5 years). Ventricular arrhythmias are cardiac rhythm disturbances that can trigger ventricular fibrillation and sudden cardiac death (SCD). SCD of arrhythmic origin is responsible for up to 50% of deaths in patients with HF. In patients with HF and life-threatening ventricular arrhythmias, implantation of a defibrillator or resynchronizer has been shown to reduce the incidence of SCD due to ventricular arrhythmias and ventricular fibrillation. However, these devices are very expensive and cause significant adverse effects (risk of infection, inappropriate shocks, increased mortality with the number of shocks, etc.) that limit patients' quality of life.

[0007] In patients with HF, there is a modification of the electrical properties of cardiomyocytes, which consists of a decrease in both Na and K currents with internal rectification (l Na he K i) as a consequence of the decrease in the number of Nav1.5 and Kir2.1 channels, which generate these currents, respectively. These changes produced by HF are called "electrical remodeling." Electrical remodeling promotes the development of potentially fatal ventricular arrhythmias.

[0008] Currently approved and available antiarrhythmic drugs (AADs) produce proarrhythmic effects, and these are more pronounced the higher the patient's arrhythmic risk. Thus, in patients with HF, electrical, anatomical, and histological remodeling promotes the development of arrhythmias, diminishes the effect of AADs, and increases sensitivity to their proarrhythmic effects.

[0009] Group I PFAAAs (hydroxyquinidine, procainamide, disopyramide, lidocaine, propafenone, and flecainide) inhibit l Na by blocking Nav1.5 channels and, as a result, can lead to dangerous proarrhythmic effects. In fact, they are contraindicated in patients with HF because they significantly decrease cardiac excitability in patients who already have compromised cardiac excitability due to electrical remodeling. Therefore, they increase mortality in patients with HF and arrhythmias.

[0010] Group II FFAAs (β-adrenoceptor antagonists or beta-blockers) antagonize the proarrhythmic effects of sympathetic nervous system activation and, except in patients for whom they are contraindicated (e.g., asthmatics), are used for the treatment of HF. Although they are considered part of the optimal treatment for HF according to therapeutic guidelines, mortality in patients with HF (>50% in the first 5 years after diagnosis) due to arrhythmic causes (>50% of total mortality) remains very high.

[0011] Group III anticholinergics (amiodarone, dronedarone, sotalol) prolong ventricular repolarization. Dronedarone is contraindicated in patients with HF, and sotalol is not used (unless the patient has a defibrillator) because it excessively prolongs repolarization (the QT interval on the electrocardiogram), inducing the onset of ventricular arrhythmias in patients who, as a result of electrical remodeling, already have a very prolonged QT interval. Only amiodarone is indicated, but it produces numerous and serious adverse reactions that require its withdrawal within a maximum of two years and increases mortality from non-cardiac causes.

[0012] Group IV FFAA (verapamil and diltiazem) block calcium channels and are contraindicated in patients with HF because they depress contractility.

[0013] Arms of groups I, III and IV act by inhibiting cardiac ionic currents by blocking the channels that generate them. In various cardiac pathologies, it would be necessary to be able to increase the l Na and / or the l Ki of human cardiomyocytes. This need is especially pressing in patients with HF, but is not limited to them. The decrease in l Na It depresses the excitability of the myocardium, slowing the conduction of the cardiac electrical impulse, which favors the appearance of arrhythmias. The decrease in the l Ki In addition to decreasing excitability, it prolongs cardiac repolarization, which prolongs the QT interval of the electrocardiogram, being an additional factor that also promotes the appearance of arrhythmias.

[0014] However, at the present time, there are no specific pharmacological tools available capable of increasing either the l Ki nor the l NaFrom the perspective of pharmacological treatment of life-threatening ventricular arrhythmias, patients with HF are therapeutic orphans.

[0015] Two compounds used in the treatment of type 2 diabetes (dapagliflozin and empagliflozin) have been described to reduce morbidity and mortality in patients with HF, and it is suspected that they may also have an antiarrhythmic effect (Braunwald E. Gliflozins in the Management of Cardiovascular Disease. N Engl J Med. 2022;386:2024-2034). Furthermore, it has been shown that incubating healthy human cardiomyocytes with these compounds increases Na+ current. + and K + (Dago, M. et al. Empagliflozin and Dapagliflozin Increase Na+ and Inward Rectifier K + Current Densities in Human Cardiomyocytes Derived from Induced Pluripotent Stem Cells (hiPSC-CMs) Cells 2022;11:3707). The authors propose that increased l Naand IKI could contribute to the cardioprotective effect.

[0016] EXPLANATION OF THE INVENTION

[0017] Peptides for the treatment of heart disease.

[0018] Given the need to find a new therapeutic approach to treat cardiac diseases such as ventricular arrhythmias in patients with HF, among others, it was decided to further study the cardiac sodium channel Nav1.5, encoded by the SCN5A gene. Analysis of the protein has allowed us to identify a nuclear localization signal (NLS) at the N-terminus of Nav1.5 (NCBI protein sequence reference number: NP_000326.2). This signal is located between amino acids 13 and 37 of Nav1.5 (SEQ ID NO: 1) and is included in a 132-amino acid peptide described by Matamoros, M. et al. (Nav1.5 N-terminal domain binding to α1-syntrophin increases membrane density of human Kir2.1, Kir2.2 and Nav1.5 channels. Cardiovasc Res. 2016;110:279-290). This 132 amino acid peptide also contains an α1-syntrophin binding site and exerts a chaperone-like effect by increasing the density of Nav1.5, Kir2.1 and Kir2.2 channels in the cytoplasmic membrane.

[0019] In order to modulate the expression of SCN5A and the gene encoding Kir2.1 (KCNJ2), peptides have been designed that maintain the NLS but are smaller in size to facilitate their access to the cell nucleus. Specifically, these are peptides of 11-12 amino acids located between positions 16 and 25 or 26 (SEQ ID NO: 2), both inclusive, of Nav1.5. This amino acid sequence is R 1 - X1X2RESLAX3X4X5-R 2 , being R 1 = R, hydrogen (H), acetyl (Ac), palmitoyl (Pal), Ac- R or Pal-R, Xi= F, Y or W; X2= T, S, C or M; X3= A, G, V, L or I; X4= I, G, A, V or L and X5= E or D and R 2 = OH, NH2, K-NH2, K(SF)-NH2, or KR-NH2, where SF refers to a fluorescent probe. Preferably, the sequence is selected from the group consisting of: SEQ ID NO: 3 (referred to internally, and also herein, as DECA-11), Pal- SEQ:ID NO: 23-NH2 and / or Pal-SEQ ID NO: 25-NH2.

[0020] When analyzing the effect of SEQ ID NO: 2, and more specifically of SEQ ID NO: 3, on the SCN5A promoter and the consequent transcription of the gene, no significant effect has been observed. However, the 11 amino acid peptide is capable of activating the transcription of KCNJ2 by ​​exerting a protranscriptional effect on the minimal promoter of the human KCNJ2 gene. This surprising result encouraged us to analyze the effects of the peptide SEQ ID NO: 2, and more specifically of SEQ ID NO: 3, on the l Na and IKI in a mouse model of IC obtaining positive results.

[0021] Another aspect of the invention relates to SEQ ID NO: 2, preferably SEQ ID NO: 3, Pal-SEQ:ID NO: 23-NH2 and Pal-SEQ ID NO: 25-NH2, for use as a medicament, more specifically for use as a medicament in heart diseases, including: ventricular arrhythmias associated with cardiac hypertrophy, heart failure and hereditary syndromes secondary to mutations in the SCN5A or KCNJ2 genes that cause loss of function of the Nav1.5 or Kir2.1 channels, such as, for example, Brugada Syndrome, Progressive cardiac conduction defect syndrome or Andersen-Tawil Syndrome, among others.A third aspect of the invention relates to a DNA molecule encoding the 11 amino acid peptide, that is, to the cDNA molecules encoding the peptides of the sequence variants SEQ ID NO: 2 (SEQ ID NO: 5-19), and preferably to the cDNA molecules encoding DECA-11 (SEQ ID NO: 5), Pal-SEQ:ID NO: 23-NH2 and Pal-SEQ ID NO: 25-NH2 (SEQ ID NO: 27 and SEQ ID NO: 28).

[0022] Another aspect of the invention relates to SEQ ID NO: 5-19, 27-28, preferably SEQ ID NO: 5, SEQ ID NO:27 and SEQ ID NO:28 for use as a medicine, more specifically for use as a medicine in heart diseases, including: ventricular arrhythmias associated with cardiac hypertrophy, heart failure and hereditary syndromes secondary to mutations in the SCN5A or KCNJ2 genes that cause loss of function of the Nav1.5 or Kir2.1 channels, such as, for example, Brugada Syndrome, Progressive cardiac conduction defect syndrome or Andersen-Tawil Syndrome, among others.

[0023] The invention also relates to vectors that include any of these cDNA molecules (SEQ ID NO: 5-19, 27-28) and to eukaryotic or prokaryotic cells that contain these vectors, or any of the cDNA sequences (SEQ ID NO: 5-19, 27-28), or any of the peptide sequences derived from SEQ ID NO: 2, preferably SEQ ID NO: 3, Pal-SEQ:ID NO: 23-NH2 and Pal-SEQ ID NO: 25-NH2.

[0024] A further aspect of the invention relates to a pharmaceutical composition comprising a peptide of sequence SEQ ID NO: 2, preferably SEQ ID NO: 3, Pal-SEQ:ID NO: 23-NH2 and Pal-SEQ ID NO: 25-NH2, and a pharmaceutically acceptable excipient.

[0025] Likewise, the invention also relates to a pharmaceutical composition comprising any of the cDNA molecules defined by SEQ ID NO: 5-19, 27-28 and a pharmaceutically acceptable excipient.

[0026] Another aspect of the invention relates to pharmaceutical compositions that include vectors as described above, that is, comprising any of these cDNA molecules (SEQ ID NO: 5-19; 27-28) and a pharmaceutically acceptable excipient. It also relates to pharmaceutical compositions that include eukaryotic or prokaryotic cells that contain these vectors, or any of the cDNA sequences (SEQ ID NO: 5-19, 27-28), or any of the peptide sequences derived from SEQ ID NO: 2, preferably SEQ ID NO: 3, Pal-SEQ:ID NO: 23-NH2 and Pal-SEQ ID NO: 25-NH2, and a pharmaceutically acceptable excipient.

[0027] Another aspect of the invention relates to any of the pharmaceutical compositions described in this specification for use in the prevention and / or treatment of cardiac diseases, including: ventricular arrhythmias associated with cardiac hypertrophy, heart failure and hereditary syndromes secondary to mutations in the SCN5A or KCNJ2 genes that cause loss of function of the Nav1.5 or Kir2.1 channels, such as, for example, Brugada Syndrome, Progressive Cardiac Conduction Defect Syndrome or Andersen-Tawil Syndrome, among others.

[0028] Table 1. Sequence table

[0029] BRIEF DESCRIPTION OF THE DRAWINGS

[0030] To complement the description being made and in order to help better understand the characteristics of the invention, a set of figures is attached as an integral part of said description, in which, for illustrative and non-limiting purposes, the following has been represented:

[0031] Figure 1. Current density generated by human Nav1.5 channels as a function of the membrane potential of the test pulse in the presence or absence of two peptides of different sizes (15-25 which is DECA-11 (SEQ ID NO: 3) or 25-39 (SEQ ID NO: 4)) that form part of the N-terminal end of the Nav1.5 channel. In each panel, the peptide studied is named, describing the position of the first and last amino acids of the human Nav1.5 channel sequence. Figure 2. Current density generated by human Kir2.1 channels as a function of the membrane potential of the test pulse in the presence or absence of peptide 15-25 which is DECA-11 (SEQ ID NO: 3).

[0032] Figure 3. Density relationships of the l Na (Fig. 3A) the «i (Fig. 3B) as a function of the applied pulse membrane potential recorded in human cardiomyocytes derived from induced pluripotent stem cells.

[0033] Figure 4. Traces of l Na sustained or “beat” (lNai_) recorded in human cardiomyocytes derived from induced pluripotent stem cells in the presence (Fig. 4B) or absence (control) of DECA-11 (Fig. 4A). lNai_ was considered the sustained current sensitive to tetrodotoxin TTX (10 pM) and was quantified as a percentage of the peak current (Fig. 4C).

[0034] Figure 5. Luciferase assays to analyze the effect of DECA-11 (SEQ ID NO: 3) on the transcription of SCN5A (Fig. 5A) and KCNJ2 (Fig. 5B). Relative luminescence is expressed in arbitrary units (ALU).

[0035] Figure 6. Diagram of the protocol followed to generate the mouse model with HFpEF. Time is expressed in weeks (W). The weeks in which the baseline (ECHO1) and final (ECHO2) echocardiograms were performed before transfer to our laboratory are indicated. Animals that underwent surgery without aortic ligation are called Sham, and those that underwent aortic ligation are called TAC.

[0036] Figure 7. Capacitance (pF) of dissociated cardiomyocytes from Sham and TAC mice treated with Aav-dt-T (control) or Aav-DECA-11 (Aav-SEQ ID NO: 5).

[0037] Figure 8. iNa density-voltage relationships obtained in dissociated cardiomyocytes from Sham mice treated or not with DECA-11 (A) and TAC mice treated or not with DECA-11 (B); summary of results (C). In panel C, C stands for Control and D11 for DECA-11.

[0038] Figure 9. iKi density-voltage relationships obtained in dissociated cardiomyocytes from Sham mice treated or not with DECA-11 (A) and TAC mice treated or not with DECA-11 (B); summary of results (C). In panel C, C stands for Control and D11 for DECA-11.

[0039] Figure 10. Images of HEK293 cells treated for 24 h with Ac-SEQ ID NO: 23(SF)-NH2 and Pal-SEQ ID NO: 23(SF)-NH2 at a concentration of 10 pM obtained by fluorescence microscopy.

[0040] Figure 11. Conformational analysis by circular dichroism of Ac-SEQ ID NO: 3- NH2, AC-SEQ ID NO: 25-NH2 and Ac-SEQ ID NO: 26-NH2, in H2O and H2O / TFE mixtures (60:40), performed at 5°C and with scans between 190 and 250 nm.

[0041] Figure 12. Stability graph of Ac-SEQ ID NO: 26-NH2 in human blood serum / H2O (50:50), with incubation at 37°C and control by UPLC-MS at 24 and 72 hours.

[0042] Figure 13. iNa density-voltage relationships obtained in CHO cells expressing human Nav1.5 channels and treated or not with the Pal-SEQ ID NO: 23-NH2 peptide (indicated as D9 in this figure), at a concentration of 10 pM (A). B represents the normalized density of the maximum peak of the iNa. Na recorded in CHO cells expressing Nav1.5 channels that had been treated (white squares) or not (black circles) for 24 h with Pal-SEQ ID NO: 23-NH2, at a concentration of 10 pM. The white circles represent the normalized density of the maximum peak of the Nav1.5 channel. Na recorded in CHO cells expressing Nav1.5 channels together with the cDNA encoding the fragment containing amino acids 15-25 (DECA-11) described in SEQ ID NO: 3 (SEQ ID NO: 5).

[0043] Figure 14. INa density-voltage relationships obtained in CHO cells expressing human Nav1 .5 channels and treated or not with the Pal-SEQ ID NO: 25-NH2 peptide (D10 in this figure), at a concentration of 10 pM (A). B represents the normalized density of the maximum peak of the I Na recorded in CHO cells expressing Nav1.5 channels that had been treated (white squares) or not (black circles) for 24 h with Pal-SEQ ID NO: 25-NH2, at a concentration of 10 pM. The white circles represent the normalized density of the maximum peak of the Nav1.5 channel. Na recorded in CHO cells expressing Nav1 .5 channels together with the cDNA encoding the fragment containing amino acids 15-25 (DECA-11) described in SEQ ID NO: 3 (SEQ ID NO: 5).

[0044] PREFERRED EMBODIMENT OF THE INVENTION

[0045] The present invention is illustrated by the following examples, which are not intended to be limiting of its scope.

[0046] Example 1. Peptide design

[0047] Among other tools, in the analysis of Nav1.5 (NCBI: NP_000326.2), a nuclear localization site prediction algorithm was used (Nguyen Ba AN, et al. NLStradamus: a simple Hidden Markov Model for nuclear localization signal prediction. BMC Bioinformatics. 2009; 10:202) with which an NLS (SEQ ID NO: 1) was identified between amino acids 13 and 37, both included, of Nav1.5. Although the size of the NLS is not excessive, smaller peptides were designed seeking to identify a minimal fragment capable of accessing the nucleus of the cells and increasing the l Naand IKI - Among them, an 11 amino acid peptide (SEQ ID NO: 3) and a 15 amino acid peptide (SEQ ID NO: 4) were selected, which were generated in the laboratory. The 11 amino acid peptide was selected with a view to leaving an Arginine (R) residue at the N-terminus to facilitate peptide delamination if necessary and increase its biological stability. The sequences SEQ ID NO: 23-26 and their modifications were designed to study their ability to cross the cytoplasmic membrane, and / or preferred conformation, and / or stability in blood serum, and / or biological activity.

[0048] Example 2. Analysis of generated current density

[0049] In addition to their ability to enter the nucleus of cells, we were looking for peptides capable of increasing the current density generated by human Nav1.5 and Kir2.1 channels, so we analyzed both aspects.

[0050] Example 2.1. Current density generated by human Nav1.5 channels

[0051] To analyze the effect of the peptides designed in example 1 on the l Na and IKI, the cDNA encoding SEQ ID NO: 3 or SEQ ID NO: 4, described in SEQ ID NO: 5 and 20, respectively, were cloned into a pcDNA3.1 vector (+)® (Invitrogen) for the transfection of Chinese hamster ovary (CHO) cells, which were in turn transfected with the cDNA encoding human Nav1.5 channels (SEQ ID NO: 21). CHO cells were also co-transfected with the EBO-pcD leu 2 vector (0.5 pg), which encodes the expression of the CD8 surface antigen. The subsequent addition of anti-CD8 antibody, linked to Dynabeads M450® microspheres, allowed the identification of the transfected cells. After 48 h, an aliquot of the transfected cell suspension was placed in the chamber mounted on the stage of an inverted microscope (TMS, Nikon) and perfused at a rate of 1 ml / min with external solution (in mM): 50 NaCl, 80 CsCl, 1 CaCl2, 1.5 MgCl2, 5 HEPES and 5 glucose (pH 7.35 with CsOH). The micropipettes were obtained from borosilicate capillaries (mod. GD-1, Narishige) using a programmable horizontal stretcher (Sutter mod.P-2000) and its mouth was polished by heat in a microforge (mod. MF-83, Narishige). The micropipettes (tip resistance of <1.5 MQ) were filled with an internal solution containing (mM): NaF 10, CsF 110, CsCl 20, HEPES 10 and EGTA 10 (pH 7.35 with CsOH). The micropipettes were brought close to the cell membrane using a Narishige MO-103 micromanipulator and after applying a small suction, the portion of the membrane included in the lumen of the pipette was invaginated, forming a high resistance seal [on the order of GQ, “cell attached” configuration]. After the seal was formed, the patch membrane was ruptured by a small additional suction, allowing current recording using the “whole-cell” configuration of the “membrane patch-clamp” technique.Currents were recorded at 20–22°C using an Axopatch 200B amplifier, filtered at half the sampling rate, and stored on a computer for later analysis. Data acquisition, analysis, and pulse protocols were controlled using PCLAMP software. Capacitive current artifacts generated by pulses from 0 mV to +10 mV were recorded at 50 kHz (filtered at 10 kHz) to calculate cell capacitance, access resistance, and input impedance. The sampling rate was 50 kHz, and the data, filtered at 25 kHz, were stored on hard drives for later analysis using the CLAMPFIT application of PCLAMP software. Na was recorded by applying 50 ms pulses from a clamping potential of -120 mV to potentials between -80 and +50 mV in 5 mV increments. The peak amplitude of the l NaIt was normalized to the capacitance of each cell, thus calculating the density of the l Na (pA / pF). The density curves of the l Na -voltage were obtained by representing the density of the l Na depending on the membrane potential of the applied pulse. Figure 1 shows the density curves of the l Na -voltage in cells expressing Nav1.5 channels (black symbols) and in cells expressing Nav1.5 channels together with the cDNA encoding the fragment containing amino acids 15-25 described in SEQ ID NO: 3 (SEQ ID NO: 5) or the one encoding amino acids 25-39, described in SEQ ID NO: 4, (SEQ ID NO: 20) (white symbols). As can be seen, the fragment containing amino acids 15-25 (SEQ ID NO: 3) was able to significantly increase the density of the l Naat various membrane potentials. In contrast, the fragment containing amino acids 25-39 (SEQ ID NO: 4) did not. Each point represents the mean ± SEM of “n” experiments obtained from at least 3 different CHO cell plates. One-way ANOVA followed by Tukey's test. * P < 0.05 vs. cells transfected with Nav1.5 channels only.

[0052] Example 2.2. Current density generated by human Kir2.1 channels.

[0053] Given the results of example 2.1, in this example only the peptide described in SEQ ID NO: 3 was analyzed using its cDNA (SEQ ID NO: 5).

[0054] For this purpose, CHO cells were transfected with the cDNA encoding the human cardiac channels (Kir2.1) that generate the l Ki(Origene, USA) (SEQ ID NO: 22) by cotransfecting the cells, or not, with the cDNA described in SEQ ID NO: 5. In these experiments, the external solution that bathed the cells and the internal solution that filled the micropipettes had the following composition (mM): NaCl 136, KCl 4, CaCh 1.8, MgCh 1 , HEPES 10 and glucose 10 (pH = 7.4 with NaOH) and K-aspartate 80, KCl 50, KH2PO4 10, MgATP 3, HEPES 10, EGTA 5 (pH = 7.25 with KOH), respectively. The sampling frequency was 4 kHz and the data, filtered at a frequency of 2 kHz, were stored on hard drives for later analysis using the CLAMPFIT application of the PCLAMP program. The current ( .i) was recorded by applying 250 ms pulses from -120 to +20 mV starting from a clamping potential of -60 mV. Its density was obtained by normalizing the amplitude of the l K ¡r2.i at the end of the pulse by the capacitance of each cell (pA / pF). The density of l is shown in Figure 2 K¡r2.i as a function of the membrane potential (mV) of the pulse applied in cells transfected with the cDNA encoding the Kir2.1 channels and cotransfected or not with the cDNA encoding the peptide SEQ ID NO: 3. As in example 2.1 , each point represents the mean±SEM of “n” experiments obtained from at least 3 different CHO cell plates. One-way ANOVA followed by Tukey test. * P<0.05 vs. cells transfected only with Kir2.1 channels. Again SEQ ID NO: 3 was able to significantly increase the density of the lK¡r2.i •

[0055] Example 2.3. Current density generated in human cardiomyocytes

[0056] We infected human induced pluripotent stem cell-derived cardiomyocytes (h¡PSC-CM) with the adeno-associated virus serotype 9 Ad-td-Tomato (control) or with an adeno-associated virus serotype 9 containing the cDNA of peptide 15-25 (SEQ ID NO: 5) and td-Tomato (Aav-DECA-11; Aav-SEQ ID NO: 5). Commercial hiPSC-CM (Cell Cardiomyocytes®) were used, thawed and maintained in culture for 10 days following the protocols described by the manufacturer (Cellular Dynamics, USA). Infection with Ad-td-Tomato (red fluorescent protein) or Ad-DECA-11 was performed 48 h before proceeding with current recordings using the patch-clamp technique in the whole-cell configuration. The external solution that bathed the cardiomyocytes and the internal solution that filled the micropipettes had the following composition when the l was recorded Na(mM): NaCl 20, MgCh 1.5, CaCh 1 , CsCl 115, HEPES 5, glucose 10 and nifedipine (1 pM) (pH= 7.35 with CsOH) and NaF 10, CsF 110, EGTA 10, CsCl 20 and HEPES 10 (pH=7.35 with CsOH), respectively. To record the l Ki , the composition of the external solution was (mM): NaCl 148, MgCl21, CaCl21.8, KCl 5.4, NaH2PO40.4, HEPES 15, glucose 11 and nifedipine (5 pM) (pH= 7.35 with NaOH) and the internal solution (mM): KCl 148, MgCl21, EGTA 5, Creatine 2, Mg-ATP 5, phosphocreatine 5, HEPES 5 (pH=7.2 with KOH). Figure 3 shows that infection with the virus encoding DECA-11 is capable of significantly increasing the density of the l Na and IKI recorded in human PSC-derived cardiomyocytes. In this figure, each point represents the mean±SEM of “n” experiments / cardiomyocytes obtained from at least 3 different culture dishes. * P<0.05 vs. Ad-td-Tomato-infected cardiomyocytes. One-way ANOVA followed by Tukey test.

[0057] In patients with HF, the sustained component of the l increases Na called l Na L. This increase produces an increase in intracellular Na concentrations + which, in turn, alters the handling of Ca 2+ intracellular and prolongs the duration of action potentials, causing proarrhythmic effects. Therefore, we wanted to see if DECA-11 modified the l Na L. For this purpose, pulses of 500 ms duration from -120 to -45 mV were applied to human cardiomyocytes derived from PSCs and the l was recorded. Na and lNaL using the “external” and “internal” solutions just described. lNai_ is described as the component of the sustained current that is sensitive to tetrodotoxin (TTX) (10 pM) and was quantified as a percentage of the peak current (%). Figure 4 shows that DECA-11 does not increase l Na L.

[0058] All the data presented so far demonstrate that SEQ ID NO: 3 (and by extension any of the variations included in SEQ ID NO: 2) is capable of increasing the density of the l Na and IKI in heterologous expression systems and in human cardiomyocytes without modifying that of the l Na L.

[0059] Example 3. Protranscriptional effect of DECA-11

[0060] To study the protranscriptional effect, HEK-293 cells were seeded in 96-well plates and transfected with 300 ng of pLightSwitch-Prom luciferase vectors encoding the human minimal promoter (1000 base pairs) of the SCN5A or KCNJ2 genes (Active Motif) and cotransfected or not with the vector containing the cDNA of peptide 15-25 (SEQ ID NO: 5). 48 h after transfection, luciferase assays were performed using the LightSwitch Luciferase Assay reagent, also from Active Motif, and a Berthold luminometer. All assays were performed in triplicate and each point represents the mean of each technical triplicate. It was found that the peptide described in SEQ ID NO: 3 does not exert a significant effect on the minimal promoter of the human SCN5A gene (Figure 5), however, it does exert a protranscriptional effect on the minimal promoter of the human KCNJ2 gene, which implies that it increases the expression of Kir2.1 channels.

[0061] Example 4. Tests in a murine model of HF

[0062] A murine model of HF with reduced ejection fraction (HFfeR) was used to analyze the effect of SEQ ID NO: 3 on the decrease in l Na and the l Ki produced by the electrical remodeling associated with HF. These mice underwent transverse aortic constriction (TAC) by ligation; the pressure overload caused cardiac hypertrophy and HF. The mice were generated at the Medical Research Center of the University of Navarra (CIMA), under the direction of Dr. Aránzazu González Miqueo.

[0063] Eight-week-old mice were administered 3.5x10 10adeno-associated virus type 9 particles encoding either the fluorescent reporter td-Tomato (Aav-dt-T) alone or the reporter and the DECA-11 peptide. Echocardiographic monitoring was performed at 12 weeks of age (baseline), and at week 13, the animals underwent surgery with aortic ligation (TAC animals) or without ligation (Sham animals). The upper part of Figure 6 shows each of the procedures the animals underwent according to their age (in weeks); the lower part shows the four groups of animals that underwent surgery with ligation (TAC) or without ligation (Sham animals).

[0064] At week 19 of life, a new echocardiographic (ECHO) follow-up study demonstrated that the TAC mice had HFpEF, as expected. That same week, they were sent to the UCM animal facility. The mice were then heparinized and anesthetized, then euthanized by cervical dislocation, one per day. A thoracotomy was performed to extract the heart, which was mounted through the aorta in a cannula connected to a Langerdorff apparatus for enzymatic dissociation of cardiac myocytes. After heart extraction, it was cannulated through the aorta and placed in a Langerdorff apparatus. First, the hearts were perfused with a modified Tyrode's solution (composition in mM: NaCl 136, KCl 4, MgCh 2, CaCh 1.8, HEPES 10, glucose 10, pyruvic acid 3 and thiamine 0.1, pH=7.4 with NaOH) bubbled with oxygen at 37° C for 3-5 minutes.Subsequently, they were perfused for another 8 minutes with the same solution that did not contain CaCh. After 1 or 2 minutes, the heart stopped beating. Next, they were perfused with Tyrode's solution without CaCh supplemented with 120 u / mL of Worthington collagenase type II and 0.5 u / mL of Sigma protease XIV. Finally, the hearts were perfused with a solution called KB with the following composition (mM): KCl 25, KH2PO4 10, MgCl2 2, EGTA-K 0.5, HEPES-K 5, glutamic acid 100, aspartic acid 10, taurine 20, creatine 5 and glucose 10 (pH = 7.2 with KOH). After this entire process, the heart was removed from the cannula and agitated in aliquots of KB solution where the myocytes were disaggregated and stored in the refrigerator for at least one hour before beginning the electrophysiological recordings.

[0065] Figure 7 shows the capacitance (pF) of dissociated cardiac myocytes from the four groups of mice (12 in total). Capacitance is an indirect and reliable measure of cell size. As can be seen, there were no significant differences in the capacitance of myocytes from Sham animals, regardless of whether they were infected with Ad-td-Tomato (control) or Aav-DECA-11. In contrast, the capacitance of cardiomyocytes from animals subjected to the TAC procedure was significantly greater (P<0.05) than that of control and DECA-11 Sham animals. This result is consistent with expectations, since TAC animals exhibit cardiac hypertrophy and, therefore, their cardiomyocyte sizes are larger than those of normal hearts.

[0066] The capacitance of myocytes from TAC animals treated with DECA-11 was significantly lower than that of untreated TAC animals, indicating that DECA-11 administration prevents the development of cardiomyocyte hypertrophy associated with HFpEF.

[0067] To register the l Na In mouse cardiomyocytes, the composition of the external solution was (mM): NaCl 4, MgCl21 , CaCl21 , CdCl20.1 , CsCl 133.5, HEPES 20 and glucose 11 (pH=7.35 with CsOH) and the one filling the recording micropipette (mM): NaF 10, CsF 110, CsCl 20, HEPES 10 and EGTA 10 (pH 7.35 with CsOH). The rest of the protocols and conditions were the same as those used for recording the l Na in human cardiomyocytes derived from PSCs (according to Example 2.3). As shown in Figure 9, in animals with HFpEF, and as a consequence of electrical remodeling, the density of the l Narecorded in cardiomyocytes of control TAC mice was significantly lower than that of control Sham mice. Pretreatment with DECA-11 increased the density of the Na of the cardiomyocytes of Sham animals and, more importantly, significantly and markedly increased the density of the l Na of TAC cardiomyocytes. This result demonstrates that the administration of DECA-11 prevents the decrease in the density of the l Na produced by ICfeR in a mouse model.

[0068] As already indicated, the l was also recorded in the cardiomyocytes of mice from all four groups. Ki■ For this purpose, the composition of the external solution was (mM): NaCl 140, MgCl21 , CaCl21 , KCl 4, HEPES 10, 4-aminopyridine 2, nifedipine (1 pM), atropine (0.1 pM), glibenclamide (10 pM) and glucose 10, (pH= 7.35 with NaOH) and the internal solution (mM): K-aspartate 80, KCl 50, KH2PO410, MgATP 3, HEPES 10, EGTA 5 (pH=7.25 with KOH). As a consequence of the electrical remodeling associated with HFfeR produced by the transverse constriction of the aorta, the density of the l Ki was significantly lower in the cardiomyocytes of control TAC animals than in control Sham animals (Figures 9A and 9B). In both Sham and TAC animals, administration of DECA-11 significantly increases the l K i. In fact, the density of the l Ki In TAC animals pretreated with DECA-11 is not different from that recorded in Sham animals. These results demonstrate that the administration of DECA-11 prevents the decrease in the density of the l Kiproduced by ICfeR in a mouse model.

[0069] Example 5. Solid-phase synthesis of DECA-11 analogs

[0070] The synthesis was performed manually using polypropylene syringes, with variable volume depending on the amount of resin used, equipped with a porous polyethylene filter plate. Generally, the resin was kept in the syringe by adding the appropriate reagents and solvents in each case so that it remained covered and solvated. The polymeric support was agitated with an orbital shaker. Excess reagents, solvents, and possible byproducts were removed by filtration. A TentaGel resin with a Rink amide spacer with a loading of 0.24 mmol / g was used. For the elongation of linear peptides, an Fmoc^Bu strategy was employed, following the protocol described in Table 2.

[0071] Table 2. General protocol for deprotection and coupling of amino acids aOnly in cases with a positive ninhydña assay at stage 6. If the assay was positive at stage 9, stages 7-9 were repeated.

[0072] Once the peptide elongation is complete, the protocol to follow for the acetylation of the N-terminal end is described below in Table 3.

[0073] Table 3. General protocol for acetylation of amines at the N-terminal end.

[0074] For / V-palmitoylated derivatives, once the peptide elongation is complete, the general palmitic acid coupling protocol is described in Table 4.

[0075] Table 4. General protocol for coupling palmitic acid at the / V-terminal end.

[0076] For the cleavage of the products from the resin, the acidolytic mixture consisting of TFA:EDT:H2O:TIPS in proportions 94:2.5:2.5:1 was added to the resin placed in the syringe (approximately 1 ml of solution per 100 mg of resin). After the addition of the solution, the reaction was allowed to take place at room temperature for 3 hours. Subsequently, the filtrates were collected in a Falcon tube and cold ethyl ether was added to precipitate the peptide. The mixture was centrifuged for 10 min at 5000 rpm and at -15 °C. The supernatant was decanted and the process was repeated two more times. The dry precipitate obtained was dissolved in water or water / acetonite mixtures and lyophilized to obtain the final crude oil.

[0077] The crude oils were analyzed by reversed-phase HPLC-MS using a SunFire C18 column (2.1 mm x 50 mm; 3.5 pm). A gradient of CH3CN + 0.1% formic acid and H2O + 0.1% formic acid was used, at a flow rate of 1 ml / min and with continuous detection at a wavelength between 230-400 nm.

[0078] The reaction crudes were purified by flash chromatography with Biotage Sfár Bio C18 reversed-phase cartridges, using a gradient of CH3CN + 0.05% in H2O + 0.05% TFA as solvent. The collected fractions were analyzed by reversed-phase HPLC using an Eclipse Plus C18 column (4.6 mm x 150 mm, 5 pm), a gradient of CH3CN and H2O + 0.05% TFA, at a flow rate of 1.5 ml / min and with detection at 220 nm wavelength. The characterization of the synthesized peptides is shown in Table 5.

[0079] Table 5. Characterization of the synthesized peptides. b Gradient: 5-95% (MeCN + 0.1% formic acid / H2O + 0.1% formic acid), 10 minc Gradient: 20-95% (MeCN + 0.1% formic acid / H2O + 0.1% formic acid), 10 min d Gradient: 2-95% (MeCN + 0.1% formic acid / H2O + 0.1% formic acid), 10 min

[0080] Example 6. Studies of the access to the cellular interior of the peptides Ac-SEQ ID NO: 23(SF)-NH2 and Pal-SEQ ID NO: 23(SF)-NH2

[0081] In this set of experiments, the ability of various DECA11-derived peptides (SEQ ID NO: 3) conjugated to the fluorescent probe fluorescein-thiourea to enter the cell interior has been analyzed. For this purpose, an imaging study was performed using fluorescence microscopy in HEK293 cells. In this way, if the peptides are able to cross the cell membrane by themselves and only if they cross it, they will generate a fluorescent signal that can be detected by fluorescence microscopy. The cells, grown at 37 ° C in Dulbecco's modified Eagle's medium (DMEM) together with 10% fetal bovine serum, 100 U / mL of penicillin, and 100 pg / mL of streptomycin, were incubated for 24 h with Ac-SEQ ID NO: 23(SF)-NH2O Pal-SEQ ID NO: 23(SF)-NH2 at a concentration of 10 pM. After the incubation period, the cells were seeded on polylysine-coated glass coverslips placed inside 35 mm dishes and left to rest for 24 h.Cells were then fixed by incubating with 4% paraformaldehyde for 15 minutes. After this time, cells were treated with 4',6-diamidino-2-phenylindole (DAPI, 300 nM), a fluorescent dye that binds to adenine- and thymine-rich regions of DNA and is therefore used to label cell nuclei. Finally, cells were incubated with PROLONG mounting medium (4 pL) and kept at rest at 4°C overnight. The following day, images were obtained using a THUNDER imager 3D Tissue fluorescence microscope.

[0082] The comparative study of the permeability of the fluorescent peptides Ac-SEQ ID NO: 23(SF)-NH2 and Pal-SEQ ID NO: 23(SF)-NH2 is shown in Figure 10. It can be seen that the peptide Ac-SEQ ID NO: 23(SF)-NH2 is not able to penetrate the interior of the cells in culture, so no green fluorescent signal is recorded. On the contrary, the peptide Pal-SEQ ID NO: 23(SF)-NH2, with an N-terminal Pal group, showed good ability to penetrate the cell interior.

[0083] Example 7. Conformational studies using circular dichroism

[0084] Circular dichroism spectra were recorded in aqueous solution and in a 60:40 H2O:trifluoroethanol (TFE) mixture. The concentration used was 10 pM and the temperature (T) was set to 5°C. Measurements were made between 190 and 250 nm using a 0.1 cm pathlength cell. Experiments were performed at a scan rate of 50 nm / min, a response time of 2 s, and a bandwidth of 1 nm. The spectra obtained are the average of 4 baseline-corrected scans. Helicity was calculated using equation [0] 222 / [0]max, where [0] m áx= (- 44000+250T) (1- / n). The finite length correction ( ) was taken as k = 4, while n refers to the number of peptide residues. The helicity values ​​of the peptides studied are shown in Table 6, while the circular dichroism spectra are shown in Figure 11.

[0085] Table 6. Helicity observed by circular dichroism for the peptides studied. From the values ​​in Table 6 and the graphs in Fig. 11 , it can be deduced that the peptides studied show helicities between 2 and 10% in aqueous solution, which increase significantly in the presence of a helicogenic solvent such as TFE. The increase in helicity in H2O when comparing the peptide Ac-SEQ ID NO: 25-NH2 with Ac-SEQ ID NO: 3-NH2, of 8%, demonstrates that the incorporation of a K residue at the C-terminal end induces a significant increase in the structuring of the peptide into an α-helix. The incorporation of an R residue after the K in the peptide Ac-SEQ ID NO: 26-NH2 does not have a significant effect on the helicity with respect to Ac-SEQ ID NO: 25-NH2. Circular dichroism data in aqueous solution and H2O:TFE mixture reliably demonstrate that these peptides have a tendency to structure in α-helix conformations, favored by the incorporation of basic residues at the C-terminal end.

[0086] Example 8. Stability study in blood serum

[0087] The stability study of the peptide Ac-SEQ ID NO: 26-NH2 in human blood serum is shown in Figure 12. A 1 mM solution of the peptide in water was prepared and 0.5 mL was taken and mixed with 0.5 mL of human serum. The resulting solution was incubated for 72 h at 37 °C. After 24 h and 72 h, 120 pL aliquots were taken and treated with 20 pL of trifluoroacetic acid to precipitate the proteins. After 30 minutes at 4 °C, the mixture was centrifuged at 13000 g and 4 °C. The supernatant, once filtered, was analyzed by UPLC-MS using a C18 Bridge column (3.0 mm x 50 mm, 2.5 pm) and a gradient of 15 and 50% CH3CN (0.1% formic acid) in H2O (0.1% formic acid) in 5 minutes.

[0088] As shown in Figure 12, 82% of the Ac-SEQ ID NO: 26-NH2 peptide remained unchanged after 24 h of incubation. Considering that the serum used was at a concentration of 50% and the percentages of unchanged peptide at different times, the half-life of Ac-SEQ ID NO: 26-NH2 was estimated at 27 h. This stability value in serum is very suitable for its potential application in the aforementioned indications.

[0089] Example 9. Effects of Pal-SEQ ID NO: 23-NH2 and Pal-SEQ ID NO: 25-NH2 on INa.

[0090] To analyze the effect of S Pal-SEQ ID NO: 23-NH2 and Pal-SEQ ID NO: 25-NH2 on the l Na generated by human cardiac Nav1.5 channels, a protocol similar to that described in example 2.1 was used. CHO cells were transfected with the cDNA encoding human Nav1.5 channels (SEQ ID NO: 21) and the l NaIt was recorded using the patch-clamp technique in its whole-cell configuration using the external and internal solutions whose composition is described in example 2.1.

[0091] In Figure 13, the density curves of the l are represented in panel A. Na -voltage recorded in CHO cells expressing Nav1.5 channels and incubated (white symbols) or not (black symbols) for 24 hours with the peptide described in Pal-SEQ ID NO: 23-NH2 (called D9 in the figure) at a concentration of 10 pM. As can be seen, the peptide described in Pal-SEQ ID NO: 23-NH2 was able to statistically significantly increase the l Naat various membrane potentials. Each point represents the mean ± SEM of “n” experiments obtained from at least 3 different CHO cell plates. One-way ANOVA followed by Tukey's test. * P < 0.05 vs. untreated control cells. The normalized peak density of the l is shown in panel B of Figure 13. Na recorded in CHO cells expressing Nav1.5 channels that had been treated (white squares) or not (black circles) for 24 h with the peptide SEQ ID NO: 25 at a concentration of 10 pM. The white circles represent the normalized density of the maximum peak of the Nav1.5 channel. Narecorded in CHO cells expressing Nav1.5 channels together with the cDNA encoding the fragment containing amino acids 15-25 described in SEQ ID NO: 3 (SEQ ID NO: 5). As can be seen, the increase produced by the Pal-SEQ ID NO: 23-NH2 peptide is similar to that produced by the cDNA encoding the fragment containing amino acids 15-25 described in SEQ ID NO: 3 (SEQ ID NO: 5). In this figure, each point represents an experiment and the bars the mean±SEM of “n” experiments obtained from at least 3 different CHO cell plates.

[0092] In Figure 14, the density curves of the l are represented in panel A. Na-voltage recorded in CHO cells expressing Nav1.5 channels and incubated (white symbols) or not (black symbols) for 24 hours with the peptide described in Pal-SEQ ID NO: 25-NH2 (called D10 in the figure) at a concentration of 10 pM. As can be seen, the peptide Pal-SEQ ID NO: 25-NH2 was able to statistically significantly increase the l Na at various membrane potentials. Each point represents the mean ± SEM of “n” experiments obtained from at least 3 different CHO cell plates. One-way ANOVA followed by Tukey test. * P < 0.05 vs. untreated control cells. The normalized peak density of the l Narecorded in CHO cells expressing Nav1.5 channels that had been treated (white squares) or not (black circles) for 24 h with the peptide SEQ ID NO: 29, at a concentration of 10 pM. The white circles represent the normalized density of the maximum peak of the Nav1.5 channel. Na recorded in CHO cells expressing Nav1.5 channels together with the cDNA encoding the fragment containing amino acids 15-25 described in SEQ ID NO: 3 (i.e., SEQ ID NO: 5). As can be seen, the increase produced by the Pal-SEQ ID NO: 25-NH2 peptide is similar to that produced by the cDNA encoding the fragment containing amino acids 15-25 described in SEQ ID NO: 3 (i.e., SEQ ID NO: 5). In this figure, each point represents an experiment and the bars the mean±SEM of “n” experiments obtained from at least 3 different CHO cell plates.

Claims

CLAIMS 1. Peptides of general formula R 1 -XIX2RESLAX3X4X5-R 2 (R 1 -SEQ ID NO: 2-R 2 ), in which, R 1 is selected from hydrogen (H), arginine (R), acetyl (Ac), palmitoyl (Pal), Ac-R or Pal-R Xi is selected from F, Y or W, X2 is selected from T, S, C or M, X3 is selected from A, G, V, L or I, X4 is selected from I, G, A, V or L, X5 is selected between E or D, and R 2 is selected from OH, NH2, K-NH2, K(SF)-NH2, or KR-NH2, where SF indicates fluorescent probe.

2. Peptide according to claim 1, wherein R 1 is selected between H, Ac or Pal.

3. Peptide according to claim 2 wherein R 2 is selected from an alkyl OH, NH2, K-NH2, K(SF)-NH2, or KR-NH2.

4. Peptide according to any of claims 1-3, which is selected from the group consisting of SEQ ID NO: 3, Pal-SEQ ID NO: 23-NH2 and / or Pal-SEQ ID NO: 25-NH2.

5. Peptide according to any of claims 1-4 for use as a medicament.

6. Peptide according to claim 5 for use as a medicament in heart diseases.

7. Peptide according to claim 6, wherein the cardiac diseases belong to the group consisting of: ventricular arrhythmias associated with cardiac hypertrophy, heart failure and / or hereditary syndromes secondary to mutations in the SCN5A or KCNJ2 genes that cause the loss of function of the Nav1.5 or Kir2.1 channels.

8. Peptide according to claim 7, wherein the hereditary syndromes belong to the group consisting of: Brugada Syndrome, Progressive cardiac conduction defect syndrome, and Andersen-Tawil Syndrome.

9. cDNA molecule encoding the peptide described in SEQ ID NO: 3, Pal-SEQ ID NO: 25-NH2 or Pal-SEQ ID NO: 25-NH2.

10. cDNA molecule according to claim 9, consisting of SEQ ID NO: 5, SEQ ID NO: 27 or SEQ ID NO:

28. 11 cDNA molecules according to any of claims 9-10 for use as a medicament.

12. cDNA molecules according to claim 11 for use as a medicament in heart diseases.

13. cDNA molecules according to claim 12, wherein the cardiac diseases belong to the group consisting of: ventricular arrhythmias associated with cardiac hypertrophy, heart failure and / or hereditary syndromes secondary to mutations in the SCN5A or KCNJ2 genes that produce the loss of function of the Nav1.5 or Kir2.1 channels.

14. cDNA molecules according to claim 13, wherein the hereditary syndromes belong to the group consisting of: Brugada Syndrome, Progressive cardiac conduction defect syndrome, and Andersen-Tawil Syndrome.

15. Vector including a cDNA molecule defined in any of claims 9-10.

16. Eukaryotic or prokaryotic cell containing any of the peptides, cDNA molecules or vectors defined in claims 1-4, 9-10, 15.

17. Pharmaceutical composition comprising a peptide defined in any of claims 1-4 and a pharmaceutically acceptable excipient.

18. Pharmaceutical composition comprising the molecule of any of claims 9-10 and a pharmaceutically acceptable excipient.

19. Pharmaceutical composition comprising the vector, and / or the cell of any of claims 15-16 and a pharmaceutically acceptable excipient.

20. Pharmaceutical composition according to any of claims 17-19 for use in the prevention and / or treatment of heart disease.

21. Pharmaceutical composition according to claim 20, wherein the heart diseases belong to the group consisting of: ventricular arrhythmias associated with cardiac hypertrophy, heart failure and / or hereditary syndromes secondary to mutations in the SCN5A or KCNJ2 genes that cause the loss of function of the Nav1.5 or Kir2.1 channels.

22. Pharmaceutical composition according to claim 21, wherein the hereditary syndromes belong to the group consisting of: Brugada Syndrome, Progressive cardiac conduction defect syndrome, and Andersen-Tawil Syndrome.

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