WO2023062451A1

WO2023062451A1 - Pegylated tat-efsevin-ta as an antiarrhythmic agent with favorable effect on heart failure caused by arrhythmia

Info

Publication number: WO2023062451A1
Application number: PCT/IB2022/058050
Authority: WO
Inventors: Ali FARHADI ANDAR ABI; Haniyeh ABOEIMEHRIZI; Mohammadhossein MALEKZADEH
Original assignee: Farhadi Andar Abi Ali; Aboeimehrizi Haniyeh
Priority date: 2022-08-28
Filing date: 2022-08-28
Publication date: 2023-04-20

Abstract

The combination of the three substances TAT, efsevin, and TA along with PEG results in "PEGylated TAT-Efsevin-TA" compound functions as an anti-arrhythmic agent with favorable effect on heart failure caused by arrhythmia. The production steps include three phases: in silico, in vitro, and in vivo. the pegylated TAT-Efsevin-TA compound is an anti-arrhythmic agent with targeted delivery functions in heart cells due to the presence of the TA part as a targeted drug delivery agent to the heart tissue and the TAT part as a penetrating peptide into the cells and mitochondrial membrane. the in vitro and in vivo tests the PEGylated TAT-Efsevin-TA compound exerts anti-arrhythmic activity and with favorable effect on heart failure caused by arrhythmia in a targeted manner and without any reported side effects.

Description

PEGylated TAT-Efsevin-TA as an antiarrhythmic agent with favorable effect on heart failure caused by arrhythmia

The combination of the three substances TAT, efsevin, and TA along with PEG results in "PEGylated TAT-Efsevin-TA” compound functions as an anti-arrhythmic agent with favorable effect on heart failure caused by arrhythmia. The production steps include three phases: in silico, in vitro, and in vivo. the pegylated TAT-Efsevin-TA compound is an anti-arrhythmic agent with targeted delivery functions in heart cells due to the presence of the TA part as a targeted drug delivery agent to the heart tissue and the TAT part as a penetrating peptide into the cells and mitochondrial membrane. the in vitro and in vivo tests the PEGylated TAT-Efsevin-TA compound exerts anti-arrhythmic activity and with favorable effect on heart failure caused by arrhythmia in a targeted manner and without any reported side effects.

A61K 9/00 - A61N 1/362

Methods for the prediction of arrhythmias and prevention of sudden cardiac death

United States Patent 7822474

Methods and kits are provided for determining an increased likelihood of the occurrence of a cardiac arrhythmia, myocardial ischemia, congestive heart failure and other diseased conditions of the heart. The methods and kits comprise measuring serum NGF levels in a subject and detecting increases in NGF levels over baseline. The methods may further comprise initiating preventive therapy in response to a detected increase in serum NGF levels.

TREATING HEART FAILURE AND VENTRICULAR ARRHYTHMIAS

United States Patent Application 20090239940

The present invention provides methods and materials for treating heart disorders, including heart failure and arrhythmia, by enhancing SERCA2a activity. Heart cells in a subject can be treated, for example, by introducing, into the heart of the subject, an adeno-associated virus subtype 6 (AAV6) viral delivery system that includes a functional nucleic acid encoding SERCA2a. For example, the functional nucleic acid encodes a non-viral therapeutic protein, thereby treating the subject.

Treating Cardiac Arrhythmias, Heart Failure, Peripheral Artery Disease and Stroke with Cyclopentyl-Triazolo-Pyrimidine or Derivative Thereof

United States Patent Application 20110144049

The present invention relates to new methods for treating and/or preventing cardiac arrhythmias, heart failure, stroke and/or peripheral artery disease by administering a composition comprising triazolopyrimidine, or derivative or metabolite thereof. In particular, the present invention relates to methods for treating cardiac arrhythmias, heart failure, stroke and/or peripheral artery disease by administering CycloPentyl-TriazoloPyrimidine.

COMBINATION THERAPY FOR THE TREATMENT OF ARRHYTHMIAS OR HEART FAILURE

United States Patent Application 20140329755

The present invention relates to method of treating arrhythmias or heart failure comprising co-administration of a late INa inhibitor and a CAMK II inhibitor.

TRIGGERING ARRHYTHMIA EPISODES FOR HEART FAILURE AND CHRONOTROPIC INCOMPETENCE DIAGNOSIS AND MONITORING

United States Patent Application 20210386312

Techniques are disclosed for detecting arrhythmia episodes for a patient. A medical device may receive one or more sensor values indicative of motion of a patient. The medical device may determine, based at least in part on the one or more sensor values, an activity level of the patient. The medical device may determine a heart rate threshold for triggering detection of an arrhythmia episode based at least in part on the activity level of the patient. The medical device may determine whether to trigger detection of the arrhythmia episode for the patient based at least in part on comparing a heart rate of the patient with the heart rate threshold. The medical device may, in response to triggering detection of the arrhythmia episode, collect information associated with the arrhythmia episode.

The pharmaceutical project mentioned above uses a PEGylated TAT-Efsevin-TA compound as an anti-arrhythmic agent with favorable effect on heart failure caused by arrhythmia. The present invention at the same time has the ability to penetrate the heart tissue in a more targeted manner, and hence can effectively eliminate cardiac arrhythmia. The goal of this invention was to produced a fusion compound to treat arrhythmia and heart failure induced by it using "Efsevin" as an anti-arrhythmic agent and "TAT" as a peptide that can penetrate the mitochondrial membrane and "TA" as a targeted drug delivery agent to the heart tissue. Additionally, the PEG component was used in this composition to improve stability, reduce renal clearance, and enhance binding to plasma proteins. These findings demonstrated that within a few days, the rate of Ca²⁺ ion pumping from the cytoplasm to the mitochondria was increased, as did the rate of Ca²⁺ uptake in the mitochondria. Additionally, the results demonstrated that Ca²⁺ accumulation in the cytosol was inhibited during the treatment period. Moreover, no signs of severe side effects such as apoptosis and proarrhythmia were observed either in cell models or in animal models as well.

According to the World Health Organization (WHO), cardiovascular diseases (CVD) are the first cause of death worldwide, accounting for 31% of all global deaths each year. Common cardiovascular diseases include coronary artery syndrome, stroke, high blood pressure, aneurysm, cardiomyopathy, rheumatic heart disease, atrial fibrillation, congenital heart disease, endocarditis, heart failure, cardiac arrhythmia, and peripheral artery occlusion, the pathogenesis of which is different from each other. WHO predicts that cardiovascular disease will remain the number one cause of obesity and diabetes until 2030 due to aging population. Also, experts predict that cardiovascular diseases will increase exponentially over the next few years due to the long-term effects of the current COVID-19 pandemic. The dramatic growth of heart failure (HF) has recently been highlighted by the American Heart Association's heart disease and stroke statistics. According to this report, HF will increase to 46% from 2012 to 2030, resulting in more than 8 million people aged 18 years and older developing HF. In addition, half of the deaths related to cardiovascular diseases have been attributed to cardiac arrhythmia.

Despite this great importance for public health, existing drugs for HF and cardiac arrhythmia are still far from ideal, as they have dangerous side effects and cannot be prescribed for long periods of time. Especially, the treatment of cardiac arrhythmia is difficult due to major side effects of common antiarrhythmic drugs. Therefore, the main focus of cardiovascular research is to identify new and safer treatments to treat cardiac arrhythmia. Vaughan Williams Class I, III, and IV antiarrhythmic drugs act by targeting plasma membrane ion channels and suppressing ectopic signal propagation but due to their effect on the action potential of the heart and as a result the conduction speed of the heart, they are prone to proarrhythmic side effects. Therefore, there is now a clear need to develop strategies to protect the heart by new drugs in a novel way, in order to suppress the arrhythmogenic signals inside the cardiomyocytes in order to reduce the complications and mortality caused by CVD including HF and cardiac arrhythmia.

Since 2001, more than two hundred FDA approvals of protein and peptide drugs have emerged as mainstream therapies. In recent years, proteins and peptides with unique biological activity and metabolism have successfully attracted the attention of researchers as an alternative treatment for cardiovascular diseases. Compared to cell-based methods, protein therapies provide more targeted interventions by exploiting regulatory pathways, improving scalability, and potentially reducing overall cost. However, protein therapies often have limitations such as poor bioavailability, unfavorable pharmacokinetics and biodistribution problems, and off-target effects. Hydrophilicity and high molecular weight of protein drugs are the main limiting factors for their delivery at the cellular level. Selective permeability and lipophilic properties of the plasma membrane prohibit the transport of protein drugs into cells. In addition, the protein structure in the process of drug transfer should not be broken to prevent their dysfunction.

One approach to overcome these limitations is protein engineering, a strategy that may not only implement a therapeutic idea and method, but may also create the intellectual property necessary for development in patients. About half of new drugs are proteins, and many of these therapies have been developed through protein engineering. Protein engineering is a powerful approach for cardiovascular therapeutic development. The term "protein engineering" is a general term that includes many protein modification techniques, including the use of molecular display techniques for targeted delivery, structural modification of proteins to create enhanced properties such as resistance to enzymatic degradation through rational design or directed evolution, it includes the integration of proteins into polymers or other protein domains for immune evasion and many others. Develops protein, peptide, and protein engineering techniques that enable more effective and efficient delivery of small molecules, proteins, and nucleic acids across biological barriers in specific target tissues. This is a necessary and important issue because effective cardiovascular protein therapies almost certainly require targeted or localized delivery to the heart.

Compared to proteins, peptides provide more efficacy, safety and tolerability in humans, and also with the ability to penetrate the cell membrane due to their smaller size, they have been developed as promising drug candidates. According to the mentioned materials, therapeutic peptides are important in the treatment of cardiovascular diseases. Current limitations in the therapeutic agent delivery process include low drug delivery efficiency, low stability, and high toxicity. The discovery of cell-penetrating peptides (CPPs) in the early 1990s proved to be very useful due to their extraordinary multi-functional delivery properties that improved the previous limitations in the delivery of targeted agents. CPPs have been shown to facilitate the delivery of a variety of agents, including oligonucleotides, therapeutic drugs, imaging agents, and proteins ranging in size from 30 to 150 kDa. In addition, CPPs target mitochondria and across the blood-brain barrier.

Protein transduction domains (PTDs), also known as cell-penetrating peptides (CPPs), are a class of diverse peptides that typically contain 5 to 30 amino acids. Unlike many proteins, they can target intracellular proteins. Importantly, PTDs can also incorporate other biomolecules such as proteins, DNA, antibodies, and contrast agents into cells, so they have great potential as therapies. Among several carrier-mediated delivery systems, the use of CPPs as carriers for cellular delivery is considered as one of the best strategies. PTDs can also efficiently carry agents whose molecular weight is several times their molecular weight and are effective for a wide range of cell types. Trans-activator of transcription (TAT: GRKKRRQRRRPQ) from HIV-1 is the first discovered PTD, and polyarginine in it indicates the type of cationic class of this PTD. TAT has shown effective transport capability for various agents, including positively charged transport through negatively charged phospholipids on cell membranes. Studies on polyarginine (from R3 to R12) have shown that octa-arginine (R8) is the minimum sequence for cellular uptake and by increasing the number of arginine, cellular uptake can be increased. Studies on polyarginine (from R3 to R12) have shown that octa-arginine (R8) is the minimum sequence for cellular uptake and by increasing the number of arginine, cellular uptake can be increased. It was later shown that octa-arginine (8R) and nona-arginine (9R) showed higher transport compared to undeca-arginine (11R) and dodeca-arginine (12R). Energy-independent pathways, direct transfer by hydrophobic interaction between the cell membrane and hydrophobic amino acids, and endocytosis pathway are suggestions for cellular uptake. TAT peptide shows different translocation mechanisms based on its cargo, 9R mainly transports the cargo by endocytosis mechanism.

The most intractable in vivo hurdles for protein and peptide drug candidates are the immune responses resulting from protein and peptide recognition as a foreign antigen. The key factors that cause immunogenicity are impurities and aggregates, which further lead to loss of efficacy. To deal with the issue of immunogenicity, these drugs should be soluble and non-aggregating. Although the fusion protein drugs available in the market are generally safe and effective, the high cost of the drugs has become another issue for chronic patients. In order to reduce cost and side effects, drug candidates should have the potential to maintain a minimum dose. In addition, specific drug targeting at disease sites should be possible to minimize side effects and maximize efficacy. In addition, according to the development of new therapeutic peptides, special attention should be paid to improving the targeting of the ischemic zone or subcellular localization in the heart to maximize drug concentration at the site and reduce side effects. Some of these strategies are functionalization of a protein and therapeutic agent or a microcarrier or nanocarrier with a targeting sequence.

developed fusion proteins and pharmaceutical agents containing PTD for protein and agent delivery. In this method, the desired agents are combined with a PTD, also known as a cell-penetrating peptide (such as TAT). This method allows proteins and pharmaceutical agents to cross biological barriers that would normally be limited by size and charge. Peptide-drug conjugates (PDC) include simpler design, ability to interact with unknown targets, cheaper synthesis, reduced immunogenicity, and increased tissue penetration. Some of these techniques have been used to facilitate targeted delivery to the myocardium. PDCs are promising for the use of peptides in the field of therapy. PDC compounds are an emerging targeted therapy showing increased penetration and selectivity.

Many methods to deliver therapies locally to the heart require surgery such as sternotomy or thoracotomy, which inevitably involves cutting the chest wall and bones, which is very invasive for patients. The most obvious example is the "cell sheet" approach, which uses temperature-responsive polymeric templates (poly(N isopropyl acrylamide)) that undergo structural changes that cause rapid and homogeneous separation of the two-dimensional cell sheet. These cell sheets are applied directly on the damaged myocardial surfaces, which usually require the aforementioned surgical procedures. Although intramyocardial or epicardial injection can be considered non-surgical approaches for treatment, open surgery is essential for precise drug targeting that is encapsulated by biomaterials such as collagen, gelatin, fibrin, and alginate due to the dynamic motion of the heart.

Apart from direct injection into the heart tissue, several studies of alternative ways such as cardiac gene therapy using intravenous injection of adeno-associated viral vectors, ultrasound targeted microbubble destruction, catheter-based gene delivery and myocardial-infarction-specific targeted peptide conjugation to liposome have reported. Intravenously injected vehicles should not be absorbed by the glycocalyx layers present in the endothelium of blood vessels, thus increasing their half-life in the blood, but should be immediately absorbed by cardiac tissue, such as extracellular matrix (ECM)-rich. In fact, the myocardium is mostly composed of elastin and collagen. Therefore, the principle of designing delivery vehicles specific to heart tissue (myocardium) should show the characteristics of strong binding to ECM and at the same time negligible binding to the endothelium of the delivery glycocalyx. TA is one of the most abundant polyphenols and is easily found in plants such as fruits, vegetables, cocoa, etc. TA has also been used as a multifunctional coating molecule. It has attractive properties as a molecule due to its strong affinity to biomacromolecules, including DNA and proline-rich proteins, such as thrombin, gelatin, collagen, and mucin. The chemical mechanism through which TA binds to many proteins and agents includes phenolic hydroxyl-rich moieties that form multiple hydrogen bonds and hydrophobic interactions with target proteins and agents. Therefore, the strong affinity of TA to soluble proteins, elastin, collagen and ECM can increase the binding of therapeutic complexes to the myocardium of the heart tissue and allow them to remain attached to the heart tissue for a longer period of time.

Mitochondria occupy approximately 30% of cardiomyocyte volume. The role of mitochondria in oxidative metabolism, apoptosis and steroidogenesis has been well established, and mitochondrial defects play a central role in a wide range of disorders. Not surprisingly, drug targeting of mitochondria has the potential to provide therapeutic benefits in a wide range of diseases associated with mitochondrial dysfunction, including cancer, obesity, cardiovascular and neurodegenerative diseases. Recently, it has been shown that there is an important role in mitochondrial calcium uptake for rhythmic regulation of the heart. The cardiac contractile cycle begins with an influx of extracellular Ca 2+ into the heart followed by release of Ca 2+ mainly from the sarcoplasmic reticulum (SR) to initiate muscle contraction. Although mitochondria are best known for their role in providing energy to the cell, they also serve as an internal store of calcium. Abnormalities of calcium homeostasis often lead to electrical and contractile dysfunction and can cause ischemic dilated cardiomyopathy (DCM) which is one of the most common causes leading to HF, a condition in which the ventricular cavities become enlarged and impair systolic and diastolic function. Myocardial dysfunction is partially attributed to changes in the function of contractile proteins and excitation-contraction coupling (ECC).

The mitochondrial outer membrane (MOM) is the interface between the mitochondria and the cytosol, acting as an "intermediate" between the energy-producing mitochondrion and the rest of the cell. MOM controls normal metabolite and energy exchange between mitochondria and cytoplasm, and its permeability causes cell death by releasing apoptogenic factors in the cytosol. Therefore, the fine regulation of MOM permeability is crucial for cellular metabolism, and its alteration is often associated with mitochondrial dysfunction. A significant part of MOM permeability is controlled by voltage-dependent anion channel (VDAC), which is the main metabolite channel and the most abundant protein in MOM. VDAC forms the major pathway for the exchange of small ions, ATP, ADP, and other water-soluble mitochondrial metabolites across the MOM and serves as a junction for a variety of cellular signals.

Due to its central role in controlling MOM permeability and thus mitochondrial function, VDAC is a promising drug target for the treatment of a wide range of mitochondria-related pathologies. Voltage-gated anion channel 2 (VDAC2) is a large pore-forming protein (32 kDa) in the mitochondrial outer membrane that plays an important role in apoptosis by interacting with pro-apoptotic proteins such as Bcl2 family proteins and calcium signaling/homeostasis, which in cardiomyocytes is located in the vicinity of Ca2 release sites from SR. Increasing evidence shows the importance of VDAC2 in the physiological function of the heart. It has been shown that increased calcium uptake through VDAC2 reverses the arrhythmia phenotype observed in the NCX1h mutant tremblor zebrafish model and the tachycardia phenotype in mice with RYR2 mutant catecholaminergic polymorphic ventricular tachycardia. Both of these arrhythmia models have dysregulated cellular calcium signaling. Studies have shown that VDAC2 is critical for cellular calcium cycling and normal cardiac function, thereby making it a promising therapeutic target for DCM, arrhythmias, and chronic HF.

According to the above paragraphs, drug targeting of VDAC2 has significant therapeutic potential in several diseases including cancer, neurodegeneration, cardiovascular and obesity, where changing mitochondrial physiology through VDAC may be beneficial. Therefore, modulation of mitochondrial calcium uptake can serve as a new drug strategy for the treatment of cardiac arrhythmias and the resulting problems.

Efsevin is a small ester compound (dihydropyrrole carboxylic ester) and this new compound binds to VDAC2 protein. By binding to VDAC2, Efsevin increases the permeability of VDAC2 for calcium and increases the pumping speed of calcium ions from cytoplasm to mitochondria. As a result, it increases the influx of Ca2+ into the mitochondria and prevents the accumulation of calcium in the cytosol. This rhythmic cycle restores calcium ions in the cytoplasm and enables cardiac muscle cells to develop regular rhythms of contraction and relaxation. Efsevin can also correct irregular heart rhythms in mouse and human heart muscle cells. In general, efsevin treatment increases mitochondrial calcium uptake, thereby preventing the release of spontaneous intracellular Ca2+ events in cardiomyocytes, aberrant excitatory stimuli, and arrhythmias. It is interesting to note that despite the accepted role of VDAC2 in apoptosis, in case of treatment with efsevin, no effect on apoptosis has been observed. Common side effects of antiarrhythmic drugs, such as Na+, K+, and Ca2+ channel blockers, include changes in cardiac electrophysiology such as slowing of cardiac depolarization or repolarization, often expressed as a prolonged QT interval. But in the case of efsevin, it has been shown that efsevin does not affect action potential duration in human iPSC-derived cardiomyocytes and does not block hERG activity. More importantly, the effects of efsevin administration on ECG parameters such as PR interval, QT, QRS and heart rate were not observed in treated animal models. Because efsevin targets intracellular structures to suppress the generation of aberrant depolarizations and does not affect the cardiac action potential, it may be less prone to severe side effects such as the typical proarrhythmic effects seen with class I or III antiarrhythmic drugs.

Given that the EC50 of efsevin, as determined by the SR-mitochondria Ca2+ transfer assay, is approximately 2-5 μM, this rate is relatively high in the case of its use as a therapeutic drug because it causes the drug to bind to non-specific targets. Despite the strong efficacy of efsevin compound, but because efsevin lacks several essential medicinal properties such as nanomolar affinity to the target and key pharmacokinetic properties such as EC50 in micromolar range, in order to develop efsevin drug towards human treatments, several optimization steps should be done, in this case, high affinity to the target and suitable pharmacokinetics for use in human subjects should be optimized.

There are many methods for improving the characteristics of therapeutic substances and peptides, including enhancing cell permeability, increasing stability, and lowering renal clearance, which extends the half-life in blood circulation. A prolonged half-life is favorable for patient compliance and from an economic standpoint. Several studies have investigated the usage of polyethylene glycol (PEG) to decrease renal clearance and increase binding to plasma proteins like albumin. PEG is a practical and affordable choice for modification. It has been established that polyethylene glycol (PEG) is biocompatible in the body due to its high solubility, lack of toxicity and immunogenicity, flexibility, and low protein absorption.

According to the above-mentioned paragraphs, despite all the efforts made to cure arrhythmia and heart failure, no method has yet been developed that can not only affect the heart cells with a specific function but also have an efficient function with the least possible side effects. The current invention, "PEGylated TAT-Efsevin-TA as an antiarrhythmic agent with favorable effect on heart failure caused by arrhythmia," can successfully treat cardiac arrhythmia while also having the ability to penetrate the heart tissue in a more targeted manner the heart tissue. Using "TAT" as a peptide that can penetrate the mitochondrial membrane, "TA" as a targeted drug delivery agent to the heart tissue, and "Efsevin" as an anti-arrhythmic agent and improving heart failure caused by arrhythmia, the invention aims to create a fusion composition to treat arrhythmia and heart failure caused by it. Additionally, this composition uses PEG to increase stability, decrease renal clearance, and enhance binding to plasma proteins. The findings reveal that within a few days, the rate of Ca²⁺ ion pumping from the cytoplasm to the mitochondria increased, the rate of Ca²⁺ uptake by the mitochondria increased, and the accumulation of Ca²⁺ in the cytosol was prevented. During this time, neither in cell models nor in animal models, were there any indications of serious side effects like apoptosis or proarrhythmia, and no negative effects were observed during the treatment period.

Solution of problem

By simultaneously using the capabilities of "TAT" (from human immunodeficiency virus 1), "TA (tannic acid)" molecules and also using "Efsevin" as an anti-arrhythmic agent and improving heart failure, it has been made with specific function, high efficiency and minimal side effects. Due to the presence of the TA component as a targeted drug delivery agent to the heart tissue and the TAT component as a peptide that penetrates the cell and mitochondrial membrane (considering that VADC2 protein is located in the outer membrane of mitochondria), the PEGylated TAT-Efsevin-TA combination can have a targeted function in heart cells. Furthermore, the drug efsevin, which targets the VADC2 protein and acts as an anti-arrhythmic agent, can be efficient in treating arrhythmia and the heart failure that results from it. We were also able to significantly reduce the common limitations associated with the use of therapeutic agents and peptides, such as low half-life, by employing PEGylation to extend half-life, stability, and reduce renal clearance. Finally, by overcoming the limitations of conventional therapeutic approaches, the PEGylated TAT-Efsevin-TA combination can provide a much more specific and efficient performance.

How the product is made:

The next paragraphs provide an explanation of the three phases of this invention: in silico, in vitro, and in vivo.

In silico design phase: All physicochemical and structural characteristics of TAT, TA, and efsevin were initially investigated using databases. TAT was investigated using databases including PROSO DSSP, VADAR, PDB, Uniprot, ExPASy (Protparam service), NetSurfP, Mfold, and Protein-Sol from all aspects. According to these investigations, amino acids 47-57 were considered from the TAT sequence. Additionally, Efsevin and TA were thoroughly assessed in the databases IUPHAR/BPS, tdrtargets, chEBI, PubChem, LigandBox, and chemaxon. Finally, the invented combination of TAT-TA-Efsevin was designed and evaluated using software-based methods and databases such as Molecular modeling, Structure-based drug design, Structure-based virtual screening, Ligand interaction, molecular dynamics, MolAICal, PharmMapper, GLORY, livertox, which are effective tools for drug design. These software and databases also investigated the structures between the ligand and the drug as well as the pharmacokinetic and pharmacodynamics properties of the drug. Also, to identify interactions at the molecular level, protein-ligand docking was performed using AutoDock Vina.

In vitro laboratory phase: In this phase, taking into account the results of the in silico phase, the selected sequences of TAT protein, which include 47-57 amino acids and efsevin, was synthesized. The TAT peptide was then conjugated to efsevin, and TA was PEGylated using PEG. Finally, PEGylated TA was coupled to TAT-Efsevin. Dynamic light scattering (DLS), zeta potential, NMR and scanning electron microscopy (SEM) techniques were used for the final investigation and characterization of these compounds and the final combination of TAT-Efsevin-TA/PEG. H9C2 (rat cardio myoblast) cell line was utilized to demonstrate the effectiveness of the PEGylated TAT-Efsevin-TA compound's cellular uptake once the findings of these evaluations were confirmed. Imaging was carried out using a fluorescent microscope after these cells were treated at various time intervals.

The effectiveness of the inventive pharmaceutical compound was then initially examined in permeabilized HeLa cells. Ca²⁺was first depleted from intracellular stores by incubation with thapsigargin (VWR) and then HeLa cells were permeabilized with digitonin. In an intracellular-like buffer with an appropriate cell density in each well, this experiment was carried out. A luminescence counter was used to measure the Ca²⁺-stimulated optical signals at 469 nm every 0.1 seconds while the permeabilized cells were treated with the invented compound at room temperature. Based on these parameters, a score (Sdrug) was assigned to this compound. This preliminary test verified the dose-dependent effect of the PEGylated TAT-Efsevin-TA compound on enhancing mitochondrial-Ca²⁺ uptake in HeLa cells. PEGylated and non-PEGylated TAT-Efsevin-TA were incubated with human liver microsomes at 37°C, and the reaction was started by adding 1 mM NADPH to measure the stability of efesvin compounds. They were transferred to cold acetonitrile at 0, 5, 10, 30, and 60 minutes during the reaction. Following centrifugation, the supernatant was analyzed using LC-MS/MS. Additionally, this test was conducted with mouse serum at a pH of 7.4 and a temperature of 37 ° C. This procedure also demonstrates that this invented compound is more stable than efsevin alone. The more specific performance of the newly developed drug combination was examined once the effectiveness of the drug combination was confirmed. The first step was to measure the Ca²⁺ transit from the sarcoplasmic reticulum (SR) to the mitochondria. One day before the experiment, H9C2 cells were added to a 96-well plate for this purpose. Cells were stained with Rhod-2, AM, then permeabilized with digitonin to monitor mitochondrial calcium. SR Ca²⁺ release was produced by superfusion with caffeine after the invented compound was added, and mitochondrial Ca²⁺ was constantly monitored in a fluorescence plate reader. Additionally, in permeabilized H9C2 cardiomyocytes loaded with Rhod-2 AM on a 96-well fluorescent reader plate, mitochondrial Ca²⁺ uptake in response to caffeine was assessed. This procedure involved using a Ca²⁺ chelator to limit Ca²⁺ around RyR clusters. Cytosolic calcium was measured in a fluorescence plate reader to investigate the release of cytosolic Ca²⁺ in H9C2 cells. The next phase was cultivating and differentiating mouse and human embryonic stem cells (ESC). Beating mouse EBs were given TAT-Efsevin-TA treatment on the 10th differentiation day, and beating human EBs were given the same treatment on the 15th differentiation day. Beating EBs images were obtained at a speed of 30 frames per second and analyzed with motion-detection software. Additionally, EBs were loaded with fluo-4 AM in culture medium at 37°C in order to record calcium level. Confocal microscopy was used to detect the fluorescent signals. Cardiomyocytes were labeled with 3H-labeled adenine to detect [3H] cAMP accumulation and intracellular cAMP levels. Finally, in this phase, the effect of medicinal compound on apoptosis was evaluated by TUNEL assay test.

In vivo Phase: At first, studies on biodistribution were carried out using C57BL/6 mice model in this phase. Mice were injected with pegylated and non-pegylated efsevin as well as TAT-Efsevin-TA compounds. At intervals, blood samples were taken from the mice and after 24 hours, the mice were sacrificed. The release and entrance of the drug in the blood and tissues were then determined after the mice had been sacrificed. This was followed by the collection of heart, kidney, spleen, lung, and liver tissues. Doxorubicin (DOX)-induced HF mouse models were applied during the in vivo phase as well.

Eight to twelve-week-old mice were given the invented compound and electrocardiography was then carried out under anesthesia to monitor ventricular tachycardia following epinephrine/caffeine injection. Under a light anesthesia, echocardiography examination was carried out on these mice models. Additionally, action potentials were captured utilizing the perforated patch-clamp method. Male and female mice between the ages of 8 and 16 weeks were used in this study. Ventricular cardiomyocytes were extracted from these animals, and in order to visualize Ca²⁺, cardiomyocytes were loaded with Fluo-4, AM. In all measurements, the invented compound was added 2 hours before the experiment.

To avoid the effect of the cells' quality decreasing over time, all groups were measured randomly in this experiment, and the experiment was terminated when the quality of cells was decreased. Finally, the images were recorded by a confocal microscope. The cells were visually evaluated before to and following each recording, and only healthy looking cells with distinct borders, regular striations, and no membrane blebs or granularity were recorded and included in the analysis. In other groups, after sacrificing the mice, their hearts were isolated. The ventricles were utilized to separate mitochondria after cutting and isolating the atria. The Pierce Assay method was used to estimate protein concentration, and the same quantity of proteins were dissolved in a suitable solution and utilized in each experiment. Finally, imaging was done on flat-bottomed 96-well plates and calcium uptake was evaluated.

Advantage effects of invention

According to the experiments, the combination of TAT-Efsevin-TA pegylated does not have an inappropriate effect on the action potential, electrocardiography and echocardiography parameters.

The stability of pegylated TAT-Efsevin-TA compared to non-pegylated TAT-Efsevin-TA has a significant increase.

The combination of TAT-Efsevin-TA pegylated has the ability to penetrate into heart cells in vitro and in vivo and has targeted therapeutic properties for heart tissue.

According to in vivo and in vitro experiments, the combination of pegylated TAT-Efsevin-TA increases Mitochondrial Ca2+ uptake and SR-mitochondria Ca2+ transport.

The combination of pegylated TAT-Efsevin-TA does not have toxicity or side effects according to in vitro and in vivo tests.

The combination of TAT-Efsevin-TA pegylated according to the tests done in vivo and in vitro exerts anti-arrhythmic function and improves heart failure caused by arrhythmia in a targeted manner and without side effects.

: Pegylated TAT-Efsevin-TA combination chart: The stages of invention of the pegylated TAT-Efsevin-TA combination in three main phases including: 1. In silico designs. 2. Production of Pegylated TAT-Efsevin-TA Conjugate and In Vitro Evaluations. 3. In vivo evaluations have been done. Each of these phases includes steps that are listed in the form of a chart.

: The steps of making TAT-Efsevin-TA/PEG combination. In this figure, both structural formula images and 3D models related to the components of the TAT-Efsevin-TA/PEG combination are placed together.

: cellular uptake efficiency by the combination of TAT-Efsevin-TA pegylated in H9C2 cells.

: Effects of pegylated TAT-Efsevin-TA combination in vitro and in vivo. (a) Increased mitochondrial Ca2+ uptake in H9C2 permeabilized cardiomyocytes by pegylated TAT-Efsevin-TA combination. A significant increase in mitochondrial Ca2+ absorption was observed in cells treated with pegylated TAT-Efsevin-TA combination.(b) TAT-Efsevin-TA/PEG combination reduces diastolic Ca2+ waves in cardiomyocytes of rat models. After the addition of ISO, an increase in the number of Ca2+ waves per minute was shown, which was significantly reduced after simultaneous treatment with ISO and the combination of pegylated TAT-Efsevin-TA.(c) Patch clamp recordings of rat cardiomyocytes showed that Spontaneous Action Potentials were significantly reduced by TAT-Efsevin-TA/PEG combination. After the addition of ISO, an increase in diastolic action potentials (APs) was shown, which was significantly reduced after the addition of the pegylated TAT-Efsevin-TA combination. (d) Assessment of cAMP accumulation in freshly isolated hearts from mouse models. The results show the ineffectiveness of the pegylated TAT-Efsevin-TA combination on cAMP accumulation in freshly isolated hearts of mouse models. While the addition of ISO caused a significant increase in cellular cAMP, the pegylated TAT-Efsevin-TA combination had no effect on the ISO-induced increase in cellular cAMP levels.

: Effects of pegylated TAT-Efsevin-TA combination in vitro and in vivo. (a) Increased mitochondrial Ca2+ uptake in H9C2 permeabilized cardiomyocytes by pegylated TAT-Efsevin-TA combination. A significant increase in mitochondrial Ca2+ absorption was observed in cells treated with pegylated TAT-Efsevin-TA combination.(b) TAT-Efsevin-TA/PEG combination reduces diastolic Ca2+ waves in cardiomyocytes of rat models. After the addition of ISO, an increase in the number of Ca2+ waves per minute was shown, which was significantly reduced after simultaneous treatment with ISO and the combination of pegylated TAT-Efsevin-TA.(c) Patch clamp recordings of rat cardiomyocytes showed that Spontaneous Action Potentials were significantly reduced by TAT-Efsevin-TA/PEG combination. After the addition of ISO, an increase in diastolic action potentials (APs) was shown, which was significantly reduced after the addition of the pegylated TAT-Efsevin-TA combination.(d) Assessment of cAMP accumulation in freshly isolated hearts from mouse models. The results show the ineffectiveness of the pegylated TAT-Efsevin-TA combination on cAMP accumulation in freshly isolated hearts of mouse models. While the addition of ISO caused a significant increase in cellular cAMP, the pegylated TAT-Efsevin-TA combination had no effect on the ISO-induced increase in cellular cAMP levels.

Examples

In this invention, the pegylated TAT-Efsevin-TA combination is made by simultaneously using the capabilities of "TAT", "TA" and "Efsevin" molecules as an anti-arrhythmic agent and improving heart failure. Pegylated TAT-Efsevin-TA combination with anti-arrhythmic properties, due to the presence of the TA segment as a targeted drug delivery agent to the heart tissue and the TAT segment, as a peptide that penetrates into the cell and mitochondrial membrane, it can have a targeted function in heart cells, and also due to the presence of efsevin drug as an anti-arrhythmic agent and improvement of heart failure caused by arrhythmia, with targeted binding to VADC2 protein, it can be effective in arrhythmia and heart failure caused by it. Also, by using pegylation in order to increase half-life, stability and reduce renal clearance, we were able to greatly reduce the common limitations associated with the use of therapeutic agents and peptides, such as low half-life. Finally, the conjugated TAT-Efsevin-TA can provide a more specific and efficient function by removing the limitations of conventional treatment methods. The production of this product includes three phases: in silico, in vitro and in vivo

The above design is made as an anti-arrhythmic agent and heart failure improvement with specific function, high efficiency and minimal side effects. This combination of TAT-Efsevin-TA can offer a much more specific and efficient function by removing the limitations of conventional treatment methods and can be used by doctors in all medical centers.

Claims

The combination of the three substances TAT, efsevin, and TA along with PEG results in "PEGylated TAT-Efsevin-TA” compound functions as an anti-arrhythmic agent with favorable effect on heart failure caused by arrhythmia. The production steps include three phases: in silico, in vitro, and in vivo.
According to claim 1, the pegylated TAT-Efsevin-TA compound is an anti-arrhythmic agent with targeted delivery functions in heart cells due to the presence of the TA part as a targeted drug delivery agent to the heart tissue and the TAT part as a penetrating peptide into the cells and mitochondrial membrane.
According to claim 1, by using PEGylating in order to increase half-life, stability and decrease renal clearance, we are able to greatly reduce the common limitations associated with the use of therapeutic agents and peptides, such as low half-life.
According to claim 3, the stability of the PEGylated TAT-Efsevin-TA compound is significantly increased compared to the non-PEGylated TAT-Efsevin-TA compound.
According to claim 1, the PEGylated TAT-Efsevin-TA compound has the property of penetrating heart cells in vitro and in vivo and it has targeted therapeutic properties for heart tissue.
According to claim 1 and according to in vivo and in vitro experiments, the PEGylated TAT-Efsevin-TA compound increases mitochondrial Ca²⁺ uptake and SR-mitochondria Ca²⁺ transport.
According to claim 1, the PEGylated TAT-Efsevin-TA compound does not have an inappropriate effect on action potential, electrocardiography and echocardiography parameters according to the experiments.
According to claim 1, the in vitro and in vivo tests the PEGylated TAT-Efsevin-TA compound exerts anti-arrhythmic activity and with favorable effect on heart failure caused by arrhythmia in a targeted manner and without any reported side effects.