IT201600120839A1 - Cardioplegic solution with autophagy activators for the diastolic arrest of the heart during cardiac surgery - Google Patents

Description

cardioplegic solution with autophagy activators for diastolic arrest of the heart during cardiac surgery "

The proposed invention consists of a cardiopiegic solution enriched with autophagy activators to improve cardioprotection during diastolic cardiac arrest during cardiac surgery.

Over 1,500,000 cardiac surgeries are performed each year in Europe and the United States of America. During cardiac surgery (commonly referred to as "open heart" surgery), electromechanical arrest of the heart itself is often necessary with the result that a paralysis of this organ called "cardioplegia" is induced. In this context, various solutions called cardioplegic solutions are used to stop cardiac contractions (diastolic arrest) in a form that minimizes myocardial damage and makes the operating field immobile, facilitating the task of the operating surgeon. Thanks to this diastolic arrest, the heart can be stopped for several hours during the central phase of cardiac surgery. Cardioplegic solutions with a high concentration of potassium (15-20 mM) have been the basis of cardiac myocardial arrest and protection for over 30 years. Although this type of cardioplegic solutions provide acceptable performance from a purely clinical point of view, and the myocardial damage during diastolic arrest is limited in part by the induced hypothermia and the reduction of cardiomyocytic metabolic processes, an inadequate myocardial protection during cardioplegia continues today. to be one of the main causes of intra, peri and post-operative failure (1). This problem has a significant impact in terms of early and / or late mortality, as well as post-operative complications in cardiac surgery.

The main and most relevant mechanism of myocardial damage during cardioplegia is ischemic, due to the prolonged interruption of coronary blood flow. At the cellular level, severe energy stress is generated during ischemia associated with a marked reduction in ATP and acidosis levels, an increase in mitochondrial dysfunction and oxidative stress and endoplasmic reticulum stress due to an accumulation of protein aggregates, with consequent final cardiomyocyte death by necrosis or apoptosis. Further myocardial damage will occur at the end of surgery with the resumption of normal coronary perfusion due to reperfusion stress characterized by a marked intracellular increase in oxygen free radicals. Furthermore, many evidences suggest that arrest based mainly on high potassium concentrations can lead to electrolyte imbalances, possible due to the phenomenon of ventricular stunning as well as ventricular arrhythmias. High concentrations of potassium can lead to ischemic damage, endothelial damage, microvascular damage, cell death and deterioration of ventricular function out of the extracorporeal circulation (CEC). In some cases, high potassium concentrations in cardioplegic solutions can induce localized or diffuse cellular and / molecular damage linked to an increase in oxidative stress mainly affecting smooth muscle cells, endothelial cells and resident cardiac progenitors, with great detriment to global cardiac function (1).

For all these pathological processes that can occur during the cardioplegia process, especially if prolonged, it is important to find new strategies that allow an increase in cardiomyocyte protection with consequent reduction of myocardial damage. Among these possible strategies, the enrichment of cardioplegic solutions with cytoprotective compounds designed to reduce the energy, mitochondrial and endoplasmic cellular reticulum stress induced by the interruption of coronary flow and diastolic arrest, represents a potentially effective, concrete and simple possibility. However, in the light of scientific-clinical evidence, none of the current cardioplegic solutions available on the market is able to adequately protect the heart from damage generated by the phenomenon of ischemia-reperfusion, acidosis, oxidative stress and apoptotic and cell necrosis.

We hypothesized that a spermidine-enriched cardioplegic solution could represent a potentially effective strategy for reducing heart damage during cardioplegia. Spermidine is a polyamine compound normally synthesized in mammals through the action of the enzyme spermidine synthetase which catalyzes the conversion of putriscein into spermidine. It occurs at high levels in the seminal fluid. However, the systemic synthesis of spermidine decreases dramatically during aging and in the presence of metabolic disorders such as diabetes and obesity. Experimental studies have shown that exogenous administration of spermidine significantly lengthens the lifespan in lower organisms, such as yeasts and insects (2). Furthermore, recent studies have indicated that the anti-aging effects of this compound are also marked in mammals. Of interest, in a very recent study published in Nature Medicine it was discovered how the exogenous administration of spermidine protects the heart in response to stress and significantly reduces the structural and functional abnormalities that arise during the aging process (3). These protective actions of spermidine seem to be linked to its ability to activate autophagy (3). Autophagy is an intracellular degradation mechanism through which senescent or damaged proteins and organelles are incorporated by double lipid membrane vesicles called autophagosomes which then merge with lysosomes, where the sequestered proteins and organelles are degraded. Autophagy is a fundamental mechanism for the regulation of cellular homeostasis (4). Furthermore, autophagy promotes the cellular response to stress. The activation of autophagy is a fundamental protective mechanism during myocardial ischemia, where it provides, through protein degradation, energy substrates that are used for the production of ATP (5). In addition, autophagy limits mitochondrial dysfunction, through the degradation of damaged mitochondria, oxidative stress and the accumulation of protein aggregates during ischemia.

All these evidences described strongly suggest that spermidine may represent a potential important therapeutic agent for reducing myocardial damage during cardioplegia by activating the autophagic process.

On the basis of the above and the reported evidence, we propose the formulation of a new cardioplegic solution enriched with spermidine to increase cardioprotection during cardioplegia, especially if prolonged, and reduce damage from ischemia and reperfusion and those induced by potassium. The same solution solution can also be used as a priming of the CEC.

The proposed invention consists of an ionic solution composed of sodium, potassium, magnesium, calcium and bicarbonate base, which may be present in varying concentrations, and which can be associated with other ionic compounds. Nutrient compounds such as glucose, mannitol, amino acids or others can be added to the solution. Albumin or insulin can also be added. To this cardioplegic solution, spermidine must be added, which may be present in varying concentrations. Spermidine can be added alone or with other autophagy activators such as trehalose or rapamycin. Other antioxidant or cytoprotective compounds in general can be added to the solution together with spermidine. The solution with its components can be preformed or the components can be powdered and reconstituted before use in distilled water. The solution can be combined with blood to form a mixed cardioplegia with a dilution ratio that can be 5: 1, 4: 1, 3: 1, 2: 1 or 1: 1.

The solution we propose can be used in cardiac surgery procedures that involve the use of any form of extracorporeal circulation where a diastolic arrest of the heart is expected. In addition, it can be used in heart transplantation and in any cardiovascular diagnostic method or therapeutic intervention. During open heart cardiac surgery with diastolic arrest, the solution can be administered inside the coronary arteries either by anterograde route in the coronary bulb or coronary ostia or in venous or arterial graft anastomosed to coronary arteries in case of previous myocardial revascularization surgery , both retrograde through the coronary sinus. The solution can be oxygenated and administered at a variable temperature either under normothermia or mild, moderate and profound hypothermia.

References

1. Hausenloy DJ, Boston-Griffiths E, Yellon DM. Cardioprotection during cardiac surgery. Cardiovasc Res. 2012; 94 {2): 253-65.

2. Madeo F, Eisenberg T, Buttner S, Ruckenstuhl C, Kroemer G. Spermidine: a novel autophagy inducer and longevity elixir. Autophagy. 2010; 6 (1): 160-2.

3. Eisenberg T, Abdel! Atif M, Schroeder S, Primessnig U, Stekovic S, Pendi T, et al. Cardioprotection and lifespan extension by the naturai polyamine spermidine. Nat Med.

2016.

4. Levine B, Kroemer G. Autophagy in the pathogenesis of disease. Celi.

2008; 132 (1): 27-42.

5. Sciarretta S, Zhai P, Volpe M, Sadoshima J. Pharmacological modulation of autophagy during cardiac stress. J Cardiovasc Pharmacol. 2012; 60 (3): 235-41.

Claims (12)

1. A cardioplegic solution characterized in that it contains distilled water (0.01-5L) in which some or all of the following chemical compounds are dissolved which are calcium chloride (0.05-0.6 g / L), potassium chloride ( 0.05-0.6 g / L), potassium phosphate (0.005-0.3 g / L), magnesium chloride (0.025-0.6 g / L), magnesium sulfate (0.025-0.6 g / L), sodium chloride (5-10 g / L), sodium bicarbonate (0.2-0.8 g / L), sodium phosphate (0.01-0.1 g / L), with or without other ions or inorganic compounds, added with spermidine which can be added at a concentration ranging from 1 nM to 1M, maintaining a solution pH ranging between 7 and 7.8 and with a final osmolarity of 100-600 mlliosmoles. 2. A cardioplegic solution enriched with spermidine, as described in the preceding claim, characterized in that the spermidine can be added with albumin (0.1-10%) and / or insulin (0.1-200 Ul) and / or other nutritional components such as, for non-binding purposes, glucose (0.1-10 g / L), mannitol (0.1-10 g / L), pyruvate (0.1-50 mM) , malate (0.1-50 mM), succinate (0.1-50 mM), and / or amino acids such as, by way of non-binding example, Γ L-arginine (0.5-2.5 g / L ), L-taurine (0.1-1 g / L), beta-alanine (0.1-1 g / L) and / or other components. 3. A cardioplegic solution enriched with spermidine, as described in the preceding claims, characterized in that the spermidine can be added with other antioxidant agents such as, by way of non-binding example, ascorbic acid (0.005-0.6 g / L) and / or other cytoprotective compounds. 4. A cardioplegic solution enriched with spermidine, as described in the preceding claims, characterized in that the spermidine can be added with calcium channel blockers and / or adenosine and / or beta-blockers and / or other potentially cardioprotective drugs. 5. A cardioplegic solution enriched with spermidine, as described in the preceding claims, characterized in that the spermidine can be added with trehalose (concentration range 1 nM-1M) or with α, α-trehalose together with trehalase inhibitors or with an analogue of α-α-trehalosìo resistant to trehalase activity, such as lentztrealose A, B, or C, (concentration range 1 nM-1M). 6. A cardioplegic solution enriched with spermidine, as described in the preceding claims, characterized in that the spermidine can be added with rapamycin (concentration range 1 nM-1M) or other analogues of rapamycin or cyclosporine. 7. A cardioplegic solution enriched with spermidine, as described in the preceding claims, characterized in that it can be preconstituted or can be prepared before use by adding each component in the form of powder or liquid to distilled water. 8. A cardioplegic solution enriched with spermidine, as described in the preceding claims, characterized in that it can be administered as a crystalloid, colloid or diluted solution at different concentrations and added to the blood before use, in which potassium can be varied according to the use that is made of the solution, whether to induce diastolic arrest, to keep the heart protected during diastolic arrest or in general as a protective therapy in the presence of ischemic suffering. 9. A cardioplegic solution enriched with spermidine, as described in the preceding claims, characterized in that it can be used in all types of cardiac surgery requiring cardiac arrest in diastole. 10. A cardioplegic solution enriched with spermidine, as described in the preceding claims, characterized in that it can be used to preserve the donor's heart during cardiac transplant procedures. 11. A cardioplegic solution enriched with spermidine, as described in the preceding claims, characterized in that it can be administered inside the coronary arteries both antegrade in the coronary bulb and / or in the coronary ostia and / or in venous and / or graft or anastomosed to coronary arteries in case of myocardial revascularization surgery, both retrograde through the coronary sinus. 12. A cardioplegic solution enriched with spermidine, as described in the preceding claims, characterized in that it can be administered at a variable temperature, either under normothermia or mild, moderate and deep hypothermia (range 37-18 ° C).