RU2438698C1 - Water-soluble composition, possessive properties of cardio-protector - Google Patents

Abstract

FIELD: medicine, pharmaceutics. ^ SUBSTANCE: invention relates to field of medicine and chemical-pharmaceutical industry, in particular, to medication, applied in case of myocardial infarction and operations on heart in conditions of artificial blood circulation. Water-soluble composition for intravenous introduction, contains ingredients with the following component ratio in wt %: D-glucose 23.40-28.60; potassium chloride 0.14-0.16; potassium salt of L-asparaginic acid 0.81-0.99; semi-magnesium salt of L-asparagenic acid (magnesium L-aspartate) 0.72-0.88; human insulin genetically engineered (in IU/l) 54.00-66.00; dinitrosyl complex of iron (II) with glutathione 0.19-0.23; water for injections - remaining part, with solution pH 7.40.1 at 22C. ^ EFFECT: application of composition ensures limitation of myocardial infraction size, recovery of metabolic condition of heart at reperfusion, reduces injury of membranes of post-ischemic cardiomyocytes with smaller haemodinamics disorders as compared with traditionally applied medications. ^ 2 dwg, 7 tbl, 4 ex

Description

The invention relates to the field of medicine and is intended for use as a medicine for myocardial infarction and heart surgery in a cardiopulmonary bypass.

The main damaging factors of ischemia and reperfusion of the heart are changes in the energy supply of myocardial cells and the generation of reactive oxygen species (ROS) associated with impaired intracellular ionic homeostasis. Their effect on the ischemic myocardium is able to completely block oxidative phosphorylation, damage the structure of mitochondrial and plasma membranes, cause myofibril contracture, and cause a lack of restoration of blood flow and death of cardiomyocytes. These disorders dictate the need to use metabolic correctors and antioxidants during restoration of coronary blood flow to restore the energy supply of cardiomyocytes and inactivation of the resulting ROS [1]. Typically, this approach reduces irreversible damage to the myocardium, the occurrence of arrhythmias and reduces postischemic heart dysfunction [2]. Despite the achievements of modern pharmacology, the mortality of outcomes caused by irreversible ischemic and reperfusion injury of the myocardium remains high in Russia and developed countries of the world. This is primarily due to the lack of effective drugs that simultaneously have the properties of metabolic protectors, membrane stabilizers and vasodilators. Attempts are currently being made to solve this problem by using metabolic correctors or nitric oxide (NO) donors.

The use of nitric oxide (NO) and its donors in cardiology is due to the fact that NO is an endogenous myocardial product involved in the regulation of vascular tone, cardiac contractility, platelet aggregation, and protection of cells from necrosis and apoptosis [3]. Most exogenous NO donors recommended for the treatment of myocardial ischemia and acute heart failure have found use as vasodilators or platelet aggregation inhibitors [4]. These include organic nitrates (nitroglycerin, erinite, nitrosorbide), heterocyclic amides (nicorandil) and iron complexes with NO (nitroprusside). It is believed that the most important mechanism of action of the most studied NO donor, nitroglycerin, is a decrease in pre- and afterload on the heart, a decrease in the energy demand of the myocardium as a result of expansion of peripheral vessels and deposition of blood in the veins [5]. There is evidence that nitroglycerin limits platelet aggregation [6]. Its ability to reduce the size of acute myocardial infarction (MI) and membrane damage to ischemic cardiomyocytes has been experimentally confirmed [7]. These facts served as the basis for the selection of nitroglycerin as an analogue of the developed cardioprotector. Despite prolonged use in cardiology, there is no information in the available literature on its effect on the metabolic parameters of ischemic heart. The negative effects of this drug are well documented - the rapid development of tolerance (a phenomenon that does not have a clear explanation and method of correction), side effects (headache and more dangerous - collapse), the inconvenience of its use and storage [8-10]. It was not possible to demonstrate the positive effect of nitroglycerin (and prolonged nitrates) on the life prognosis of patients with acute myocardial infarction or recently undergoing myocardial infarction. So, in the studies of ISIS-4 (Fourth International Study of Infarct Survival) and GISSI-3 (Gruppo Italiano per lo Studio della Sopravvivenza nell'Infarto Miocardico), no significant positive effect of nitroglycerin on mortality and the incidence of complications in patients after acute myocardial infarction was revealed [eleven]. In the latest version of the recommendations of the European Society of Cardiology, its intravenous administration is not recommended for use in all patients with acute myocardial infarction.

To reduce metabolic disturbances in the myocardium during ischemia and reperfusion, protectors are used, which include natural heart metabolites - a “metabolic cocktail” glucose-insulin-potassium (GIC), potassium and magnesium aspartate (asparkam, panangin), succinate derivatives (mexicor), as well as synthetic inhibitors of the oxidation of free fatty acids (trimetazidine, mildronate). Their cardioprotective effect is based on improving the energy state of ischemic cardiomyocytes - supporting higher levels of ATP and phosphocreatine (FCR) under ischemic conditions, which provides better restoration of cardiac function during the resumption of blood circulation [2, 12-15]. Most of these drugs reduce the level of free fatty acids (FFAs) circulating in the blood or reduce their consumption by the myocardium [12, 13]. This limits the toxic effect of FFA on ischemic myocardium, associated with damage to the membranes, which leads to arrhythmias and suppression of heart function [14-16]. In addition, metabolic protectors increase K ⁺ uptake and prevent overload of cardiomyocytes by Ca ²⁺ and Na ^{+ ions,} and promote glucose transport into cells [15]. The consequence of this is a decrease in the excitability and conductivity of the myocardium during reperfusion, less development of reperfusion contracture, a decrease in the occurrence of arrhythmias and the development of MI [17]. Based on these data, a widely used drug, a mixture of GIC, was chosen as one of the prototypes of the developed cardioprotector. The feasibility of using GIC was confirmed by us earlier in restoring the metabolic, morphological, and functional parameters of the heart using in vitro and in vivo models of myocardial ischemia and reperfusion [18–20]. In clinical trials of various GIC formulations in patients with acute myocardial infarction, improvements in blood biochemical parameters and heart function were also noted [12]. At the same time, insufficient efficacy in the clinical outcome of the disease was revealed - a low restriction in the size of MI and a decrease in mortality in patients [21, 22]. These features of GIC, as well as other metabolic protectors, are due to the lack of vasodilation ability and antioxidant activity. In addition, epigastric discomfort, nausea, allergic reactions, and headache were noted in a number of patients [21]. These disadvantages significantly limit the use of GIC in clinical practice.

The given results of experimental and clinical studies suggest the need for the introduction of safer and more effective donors of NO and metabolic protectors in the practice of treatment of acute coronary syndrome. In this regard, the object of the present invention is to provide a water-soluble cardioprotector having the properties of a metabolic regulator, a membrane stabilizer and a vasodilator. The problem is solved by combining an iron (II) -organic complex with nitric oxide, namely, a dinitrosyl complex of iron with glutathione (DNIC-Gln), with a polyionic biologically active medium containing energy substrates and insulin.

It is fundamentally important that the organo-iron-stabilized polymer complexes with nitric oxide are solid-phase compounds, so their use as part of potential drugs requires the development of a water-soluble form. The approach used to create the drug was to use a biologically active medium for dissolving dextran-stabilized DNAJ-Gln with cardioprotective properties. Its composition includes: d-glucose, potassium and magnesium salts of l-aspartic acid and insulin. The use of such a medium reduces the potential cytotoxicity of the complex. It is known that the result of excessive bioavailability of nitric oxide is the blocking of the respiration of mitochondria and the inhibition of the cycle of tricarboxylic acids and key glycolytic flow enzymes (phosphofructokinase and glyceraldehyde-3-phosphate dehydrogenase) [23]. Under conditions of energy deficiency, this can lead to irreversible lethal damage to myocardial cells, which is excluded with the combined use of DNJ-Glt and a biologically active medium.

The created cardioprotector is a water-soluble composition (pH 7.4) containing d-glucose, potassium chloride, potassium salt of 1-aspartic acid, semi-magnesium salt of l-aspartic acid, human genetically engineered insulin, DNA-GLn and water in the following ratio of ingredients (wt.%):

d-glucose 23.40-28.60 potassium chloride 0.14-0.16 potassium salt of l-aspartic acid 0.81-0.99 semi-magnesium salt of l-aspartic acid 0.72-0.88 human genetic engineering insulin (in IU / l) 54.00-66.00 DNJ-Gln 0.19-0.23 water for injections rest

The composition of the inventive cardioprotector is original and not described in the available literature. The combination of its components is also not obvious and cannot be empirically selected. The bibliography of patents on dinitrosyl complexes of iron (II) with low molecular weight thiol-containing ligands does not contain information on the subject of the invention — the development of such a cardioprotective chemical structure based on NO donors to reduce cardiac damage during ischemia and reperfusion.

A method of preparing a cardioprotector for animal experiments.

It consists of three stages: 1) preparation of a medium with energy substrates, 2) preparation of a dinitrosyl complex of iron with glutathione, 3) obtaining a water-soluble form of cardioprotector with a given concentration of DNJ-Glt.

Preparation of medium with d-glucose, K-Mg salts of l-aspartic acid and insulin. 26.0 g of d-glucose, 150.6 mg of potassium chloride are added to 100 ml of distilled water and stirred with gentle heating (37-40 ° C) until complete dissolution. 904.0 mg of the potassium salt of l-aspartic acid and 800.5 mg of the semi-magnesium salt of l-aspartic acid are added to the resulting solution and dissolved by stirring at room temperature. The pH of the resulting clear mixture was adjusted with 2N HCl to pH 7.4 ± 0.1 at 25 ° C. The final concentration of the components is in mm: d-glucose - 1200,0; potassium chloride - 20.2; the potassium salt of l-aspartic acid is 44.0 and the semi-magnesium salt of l-aspartic acid is 23.0. A suspension of human insulin is introduced into the solution to a concentration of 60 U / L and mixed thoroughly.

Preparation of dinitrosyl iron complex with reduced glutathione. 5 g of glutathione are dissolved in 57 ml of distilled water, 305 mg of sodium nitrite is dissolved in 3 ml of distilled water. The solutions are mixed and kept at 4 ° C for 10 minutes until a pink color appears as a result of the formation of S-nitrosoglutathione. A 5% solution of dextran in citrate-phosphate buffer is prepared by dissolving 50 g of dextran (30-40 kDa) and 9 g of NaCl in 400 ml of distilled water. Next, 30 ml of 100 mM phosphate citrate buffer (pH 7.4) and then 130 ml of distilled water are added to 60 ml of this solution. The final volume of a 5% dextran solution in buffer is 220 ml. A solution of S-nitrosoglutathione is added to it, and after stirring, the pH is adjusted to 7.4 ± 0.1 with a 30% NaOH solution. To the resulting mixture was added 20 ml of distilled water with dissolved 450 mg of FeSO ₄ × 7H ₂ O and 2.5 mg of sodium citrate. The mixture turns orange due to the formation of DNA-Glt. The solution is filtered through a 5 μm Millipore filter and poured into 0.5 ml ampoules for drying under vacuum. Chemical reactions leading to the formation of DNIC:

sodium nitrite + glutathione → nitrosoglutathione;

nitrosoglutathione + FeSO ₄ + glutathione → DNIC-Glt + glutathione disulfide.

One ampoule contains 22 mg of dry matter or 1.8 μmol DNIC-Glt in terms of iron (II) in the complex.

Obtaining water-soluble forms of the drug. The drug is prepared by dissolving DNIC-Glt in a medium with d-glucose, K-Mg salts of l-aspartic acid and insulin to create a dose of 1 μmol DNIC / kg body weight.

Methods for assessing the biological activity of a cardioprotector.

An in vivo model of regional myocardial ischemia and myocardial reperfusion was used to study the properties of the drug, providing an analysis of its effect on hemodynamic parameters, limiting the size of myocardial infarction, restoring the metabolic state of the ischemic heart, and reducing damage to the membranes of cardiomyocytes.

Model of regional ischemia and myocardial reperfusion in rats in vivo. In Wistar rats anesthetized with 20% urethane (120 mg / kg intraperitoneally), tracheotomy undergoes artificial ventilation of the lungs with room air using a KTR-5 apparatus (Hugo Sacks Electronik). The jugular vein is catheterized to stain the intact area of the heart with Evans solution at the end of the experiment. The carotid artery is catheterized to record blood pressure (BP) using a Mingograph-804 (Siemens Elema). The chest is opened by longitudinal dissection of the sternum and the heart is freed from the pericardium. To create regional myocardial ischemia, the left ventricle (LV) is stitched with an atraumatic needle 5-0 under the left ear in the direction perpendicular to the major axis of the heart. In this case, the tightening of the ligature on the anterior descending coronary artery (PNA), located in the thickness of the stitched myocardium, stops the blood supply to the myocardial site; the weakening of the ligature leads to the restoration of the coronary inflow. Indicators of the acid-base balance of arterial blood (pH, pO ₂ and pCO ₂ ) throughout the experiment are monitored on an acid-base gas analyzer ABL-30 (Radiometer) and maintained at a physiological level. After the preparation, a 30-minute period of stabilization of hemodynamic parameters (initial state) follows. Then perform PNA occlusion for 40 minutes, the duration of subsequent reperfusion is 60 minutes [20].

Determining the size of myocardial infarction. At the end of the experiment, PNA is occluded to identify the risk zone (SR) and the intact region of the myocardium and 2% Evans solution (2 ml) is bolus injected into the jugular vein. Then, the heart is cut out and LV is separated, which is frozen at a temperature of -20 ° C until histochemical determination of the MI size by staining the tissue with 2,3,5-triphenyltetrazolium chloride. To do this, cross sections of about 1.5-2 mm thick are prepared from LV, which are incubated for 10 min in a 1% solution of 2,3,5-triphenyltetrazolium chloride in 0.1 M phosphate buffer (pH 7.4 at 37 ° C) . The obtained samples are scanned, the areas of MI and SP are determined by computer planimetry using the Imagecal program. After that, the sections are weighed to determine the mass of the LV. In each group, the ratios of SR / mass of LV and MI / SR in percent are calculated.

Metabolic state of the heart. At the end of the periods of stabilization of hemodynamics (initial state), ischemia and reperfusion, the tissue ZR is frozen with Wallenberger forceps, cooled in liquid nitrogen, for the subsequent determination of metabolites. Frozen myocardial tissue samples are homogenized in cold 6% HClO ₄ (10 ml / g tissue) using an Ultra-Turrax T-25 homogenizer (IKA-Labortechnik, Germany). Proteins are precipitated by centrifugation at 3000 g and a temperature of 4 ° C for 10 minutes. Supernatants neutralize 5 M K ₂ CO ₃ to pH 7.4. The KClO ₄ precipitate was separated by centrifugation under the same conditions. Protein-free extracts are stored at -20 ° C until metabolites are detected. The dry weights of the samples are determined by weighing a portion of the tissue after extraction with HClO ₄ and drying at 110 ° C overnight. Metabolites in tissue extracts are determined spectrophotometrically, their content is expressed in μmol / g dry weight.

Damage to cell membranes. Assessed by the increase in the activity of lactate dehydrogenase (LDH) and the MV fraction of creatine phosphokinase (MV-CPK) in plasma at the end of reperfusion. Blood is collected in heparinized tubes from a venous catheter in its original state (before PNA occlusion) and after an hour of reperfusion. Plasma is separated by centrifugation (2000 rpm for 10 min at 10 ° C). Enzyme activity in freshly isolated plasma is determined spectrophotometrically at λ = 340 nm using BioSystems kits and Sigma-Aldrich reagents.

Indicators of functional hemodynamics. Changes in mean arterial pressure (BP) and heart rate (HR) are recorded on line using the Mingograph-804 (Siemens Elema) during the entire experiment. The results are statistically analyzed, comparing with the initial state and changes in the control (in the absence of the drug) or under the influence of comparison drugs. Differences between groups are assessed by analysis of variance (ANOVA) and considered statistically significant at p <0.05.

Comparison drugs. Used nitroglycerin (Perlinganite) and a mixture of glucose-insulin-potassium (GIC) of the following composition: glucose 2.8 M, human genetically engineered insulin 100 IU / L, KCl 0.1 M. Nitroglycerin, being a donor of NO, provides peripheral vasodilation and has a coronary expansion effect, is able to reduce irreversible damage to postischemic cardiomyocytes. GIC is a metabolic protector, affects the processes of intracellular metabolism, limits the size of MI [12, 20, 5, 7]. Currently, both drugs are used in clinical practice in the treatment of heart damage in acute coronary syndrome.

Example 1. Limiting the size of myocardial infarction.

Testing of the cardioprotector and comparison of its effects with the action of nitroglycerin and GIC was carried out on a model of regional ischemia and myocardial reperfusion in rats in vivo. Two series of experiments with intravenous administration of drugs were performed: 1 - before PNA occlusion and 2 - after regional ischemia in the first minute of reperfusion. GIC, nitroglycerin and cardioprotector were administered intravenously at the rate of 1 ml / kg of animal weight. The dose of nitroglycerin (1 μmol / kg rat mass) was equimolar to the content of DNIC-Glt in the cardioprotector. In the control, an equal volume of saline was administered intravenously.

Table 1 The effect of intravenous administration of cardioprotector and comparison drugs on the magnitude of myocardial infarction in rats in vivo Method of drug administration Heart attack zone / risk zone,% Control (n = 22) before PNA occlusion or in the first minute of reperfusion 47.3 ± 2.1 Cardioprotector (n = 12) 32.4 ± 2.3 ^a Nitroglycerin (n = 12) before occlusion of the PNA 37.7 ± 2.7 ^a GIC (n = 12) 39.2 ± 2.2 ^ab Cardioprotector (n = 12) 29.5 ± 2.0 ^a Nitroglycerin (n = 10) on the first minute of reperfusion 40.3 ± 2.9 ^b GIC (n = 10) 36.8 ± 2.1 ^ab Data are presented as M ± m. Control - the introduction of saline. Significantly different (p <0.05) from: ^a control; ^b cardioprotector.

Histochemical analysis of LV slices after reperfusion did not reveal significant differences in the size of the SR between the groups. The average ratio of SR / LV weight (in%) for all groups was 35.9 ± 4.0%. This indicated the validity of comparing MI restriction data in the study groups.

With the introduction of drugs before occlusion of the coronary artery, a significant decrease in MI was observed compared with the control. Moreover, the ability to limit myocardial infarction decreased in the series cardioprotector> nitroglycerin> GIC. It can be seen that the size of the MI when using a cardioprotector was significantly lower than with the introduction of GIC (p <0.05). Although the introduction of the cardioprotector MI was less than when using nitroglycerin, the differences between the groups did not reach the level of statistical significance (p = 0.53).

When drugs were administered after the end of the period of regional ischemia (in the first minute of reperfusion), a significant limitation of MI compared with the control occurred under the influence of a cardioprotector and GIC. Significant differences in the limitation of MI with the administration of nitroglycerin compared with the control were not observed (p = 0.066). The average size of the MI was minimal with the introduction of a cardioprotector: 20 ± 1% below this indicator in the GIC group (p <0.05).

The results demonstrate the advantages of using a cardioprotector, especially at the stage of early reperfusion, for limiting myocardial infarction in comparison with drugs traditionally used for this purpose.

Example 2. The metabolic state of the heart during reperfusion.

At the end of reperfusion, the levels of macroergic phosphates (ATP, ADP, AMP and FCR), creatine (Cr) and end products of glycolysis (lactate and pyruvate) were determined in the hearts of the studied groups to assess the effect of drugs on myocardial metabolism. Based on these data, the energy indicators of the postischemic heart were calculated:

energy charge of cardiomyocytes - EZ = (ATP + 0.5 ADP) / ΣAN;

degree of phosphorylation of ATP and phosphocreatine - ATP / ADP,

phosphocreatine / creatine (FCR / Cr);

the ratio of lactate / pyruvate, characterizing the degree of restoration of the cytoplasmic pyridinidinucleotides [NAD ⁺ ] / [NADH] [24].

A comparison of the obtained data with the metabolic state of the heart in the initial state (before modeling ischemia and reperfusion) is given in Table 2.

table 2 The effect of intravenous administration of a cardioprotector and comparison preparations before PNA occlusion on the metabolic state of ZR at the end of reperfusion in rats in vivo. ES = (ATP + 0.5 ADP) / ΣAH ATF / ADF FCR / Cr Lactate / Pyruvate Initial state (n = 6) 0.94 ± 0.02 8.03 ± 0.06 0.65 ± 0.03 9.8 ± 0.7 Control (n = 12) 0.60 ± 0.01 ^a 1.31 ± 0.02 ^a 0.19 ± 0.02 ^a 98.6 ± 4.6 ^a Cardioprotector (n = 12) 0.76 ± 0.02 ^{a b} 1.84 ± 0.04 ^{a b} 0.36 ± 0.03 ^{a b} 72.3 ± 4.6 ^{a b} GIC (n = 12) 0.67 ± 0.03 ^ab 1.32 ± 0.03 ^av 0.22 ± 0.03 ^av 87.4 ± 3.1 ^a Nitroglycerin (n = 12) 0.71 ± 0.02 ^ab 1.44 ± 0.03 ^ab 0.25 ± 0.02 ^ab 78.8 ± 3.9 ^ab Data are presented as M ± m. Control - the introduction of saline. Significantly different (p <0.05) from: ^a initial state; ^b control; ^{in a} cardioprotector.

From the data presented in Table 2, it can be seen that in the control, the energy state of ZR at the end of reperfusion was significantly reduced compared with the initial state of the heart. This is indicated by lower values of EZ and the ratios of ATP / ADP and FCR / Cr and more than an order of magnitude increased ratio of lactate / pyruvate. The introduction of a cardioprotector or both comparison drugs significantly improved the majority of indicators of the metabolic state of ZR, bringing them closer to the values in the initial state. An exception was the lactate / pyruvate ratio in the GIC group, which did not statistically significantly differ from the value in the control. From the analysis of indicators, it follows that the restoration of the metabolic status of SR after drug administration improved in the series GIC <nitroglycerin <cardioprotector and corresponded to their ability to limit the size of MI (Table 1). Moreover, under the influence of the introduction of a cardioprotector, the restoration of cardiomyocyte ES was significantly better than in the GIC group, and the ATP / ADP and FCR / Cr ratios were significantly higher than after using any of the comparison drugs. The obtained results indicate that the use of a cardioprotector provided a more effective restoration of aerobic metabolism in the SR during reperfusion.

Example 3. Damage to the membranes of cardiomyocytes.

Markers of damage to the sarcolemma of postischemic cardiomyocytes were the activity of lactate dehydrogenase (LDH) and the MV fraction of creatine phosphokinase (MV-CPK) in plasma. A comparison of the increase in the activity of both enzymes in the studied groups at the end of reperfusion in comparison with the values in the initial state is presented in Table 3.

Table 3 The effect of intravenous administration of a cardioprotector and comparison preparations before PNA occlusion on the activity of LDH and MV-CPK in rat plasma in vivo. The initial state End of reperfusion LDH, ME / L Control (n = 12) 71.2 ± 7.7 905.9 ± 46.0 Cardioprotector (n = 12) 72.9 ± 6.9 656.3 ± 26.2 ^a Nitroglycerin (n = 12) 70.3 ± 5.2 750.0 ± 31.6 ^{a b} GIC (n = 12) 72.9 ± 7.5 746.1 ± 35.2 ^{a b} MV-KFK, ME / l Control (n = 10) 430.2 ± 26.0 1868.0 ± 64.3 Cardioprotector (n = 12) 428.3 ± 20.1 1130.1 ± 47.2 ^a Nitroglycerin (n = 10) 429.0 ± 34.8 1323.8 ± 51.7 ^{a b} GIC (n = 10) 425.2 ± 24.5 1556.8 ± 60.3 ^{a b} Data are presented as M ± m for a series of 10-12 experiments. Significantly different (p <0.05) from: control ^a ; cardioprotector ^b .

In the initial state, the activity of LDH in plasma was almost the same in all the studied groups (Table 3). The development of MI was accompanied by a significant (12-fold) increase in the activity of LDH in plasma by the end of reperfusion in the control compared to the initial value. The introduction of a cardioprotector, as well as comparison drugs, before regional ischemia significantly reduced the release of the enzyme into the bloodstream. At the same time, a minimal increase in LDH activity was found when using a cardioprotector. It was significantly lower than after the introduction of GIC and nitroglycerin, and amounted to about 70% of the value in the control.

In the initial state, no significant differences were found between the groups in the activity of MV-CPK. By the end of reperfusion, the activity of MV-CPK in the control increased by more than 4 times compared to the initial value. A significant decrease in the activity of MV-CPK in plasma at the end of reperfusion compared with this parameter in the control occurred under the influence of all drugs. However, in the case of the introduction of a cardioprotector, the enzyme activity was lower by 15 and 28% than in the nitroglycerin and GIC groups, respectively (p <0.05).

Thus, the use of both markers of damage to cell membranes (LDH and a more specific one - MV-CPK) unambiguously indicates greater stability of the sarcolemma of postischemic cardiomyocytes in the case of cardioprotector of the proposed composition.

Example 4. Changes in hemodynamics in rats under regional ischemia and reperfusion of the heart.

The effect of the drugs on the functional hemodynamic parameters in rats in vivo was studied when they were administered before occlusion of the coronary artery. Drugs and saline were administered as indicated in the section "Limiting the size of myocardial infarction." The results are presented in figure 1 and in table.4.

When analyzing the dynamics of blood pressure, it was revealed: 1) the absence of a vasadilatory response after the introduction of GIC as opposed to cardioprotector and nitroglycerin, 2) a less sharp drop in blood pressure. in the first phase of action compared with nitroglycerin, 3) the ability of the cardioprotector to a prolonged hypotensive effect in combination with the complete restoration of blood pressure. to the end of reperfusion (figure 1).

When analyzing heart rate changes, it was found: 1) a significantly smaller decrease in this indicator with the introduction of a cardioprotector during occlusion of the coronary artery than in the case of nitroglycerin, 2) a significant decrease in the duration of arrhythmias (on average 3 times) during regional ischemia compared with both drugs comparison (table 4).

Table 4 The effect of intravenous administration of nitroglycerin, GIC and cardioprotector before PNA occlusion on heart rate and duration of rhythm disturbances during ischemia and cardiac reperfusion in anesthetized rats in vivo. Heart rate, beats / min The initial state 1st min of occlusion of the PNA 60th min reperfusion Duration of arrhythmias, sec The control 224 ± 6 228 ± 4 229 ± 6 445 ± 30 Nitroglycerine 222 ± 5 183 ± 4 ^a 227 ± 7 482 ± 42 Geek 230 ± 5 225 ± 6 224 ± 4 518 ± 51 Cardioprotector 226 ± 7 211 ± 5 ^{a b} 222 ± 5 205 ± 13 ^{a b} M ± m are presented for a series of 10 experiments. Significantly different (p <0.05) from: initial state and control ^a ; GIC and nitroglycerin ^b .

Thus, a comparison of the effects of comparison drugs with the effect of cardioprotector on hemodynamic parameters in rats in vivo indicates the advantages of using the latter in regional myocardial ischemia and subsequent reperfusion of ZR. They are less hemodynamic disturbances while maintaining a prolonged vasodilation effect and a decrease in the duration of arrhythmias. Taken together, the results indicate the promise of using a cardioprotector of the specified composition to reduce ischemic and reperfusion damage to the myocardium.

Variation of concentrations of cardioprotective components.

Two modified cardioprotective compositions were tested, containing lower and higher concentrations of the main active ingredients - d-glucose, potassium and semi-magnesium salts of l-aspartic acid, DNIC-Gln. The final pH of the cardioprotectors was unnamed and amounted to 7.4 ± 0.1 at 22 ° C (Table 5). The effectiveness of these drugs was evaluated on a regional MI model and in vivo reperfusion in rats by limiting the size of myocardial infarction while measuring hemodynamic parameters and the activity of the MV-CPK fraction in plasma.

Table 5 The composition of cardioprotectors with a modified content of components. Component Content cardioprotector-1 cardioprotector-2 d-glucose 12.0 30,0 Potassium chloride 0.10 0.20 Potassium salt of l-aspartic acid 0.36 2.25 Semi-magnesium salt of l-aspartic acid 0.32 2.0 Human Genetic Engineering Insulin 30,0 120.0 Dinitrosyl complex of iron (II) with glutathione 0.85 0.52 Water 86.37 65.03 Final pH (22 ° C) 7.4 ± 0.1 7.4 ± 0.1 The content of the components is indicated in mass%, insulin in IU / l.

The data in Table 6 show that in a series of experiments with a cardioprotector-1 and cardioprotector-2, the values of the ratio of LV / LV weight (in%) did not differ from those in the control and in experiments with a cardioprotector of optimal composition. A decrease, as well as an increase in the content of the main active components, increased the IM / RR ratio (in%) by 1.2 and 1.3 times, respectively, compared with this indicator when using a cardioprotector of optimal composition. In both cases, this led to the absence of a significant decrease in the size of myocardial infarction compared with the control, that is, to the loss of the protective effect.

Table 6 The effect of changes in the composition of the components of the drug on the limitation of the size of myocardial infarction in rats in vivo. Method of drug administration SR / LV weight,% IM / SP,% Control (n = 12) 34.0 ± 3.3 47.3 ± 2.1 Cardioprotector of optimal composition (n = 12) before occlusion of the PNA 35.5 ± 3.7 32.4 ± 2.3 ^a Cardioprotector-1 (n = 8) 34.6 ± 3.8 39.8 ± 3.4 ^b Cardioprotector-2 (n = 8) 37.0 ± 4.0 42.1 ± 4.1 ^b Data are presented as M ± m. Control - the introduction of saline. Significantly different (p <0.05) from: ^a control; ^b cardioprotector.

Figure 2 shows the changes in ADS. in rats during ischemia and reperfusion of the heart with the introduction of cardioprotector-1 and cardioprotector-2. It can be seen that a 2.5-fold decrease in the DNA-GLT content in cardioprotector-1 compared to the optimal composition cardioprotector significantly reduced the vasodilation effect of the drug in the first phase after administration and reversed its prolonged hypotensive effect. In this case, ADSr. restored to its original value at an early stage of reperfusion and did not significantly differ from the values in the control, that is, with the introduction of saline. Such a dynamic ADsr. indicated an insufficient release of NO from DNIC and the absence of complex binding to plasma proteins and red blood cells [25]. On the contrary, with the introduction of cardioprotector-2 with a 2.5-fold increase in the content of DNA-Glt during the first minutes, a sharp decrease in blood pressure occurred. up to 59 ± 3% of the initial level. Towards the end of regional ischemia ADsr. remained reduced to 68 ± 4% and practically did not recover in the future, accounting for 79 ± 4% of the initial level at the end of reperfusion. Obviously, the release of an excess of NO from DNIC-Glt could be the cause of fatal damage to cardiomyocytes at risk [23] and, as a result, an increase in infarction size (Table 6).

In a series of experiments with the introduction of cardioprotector-1 and cardioprotector-2, no decrease in the duration of arrhythmias during regional ischemia was found. The duration of rhythm disturbances did not significantly differ from the control value and amounted to 498 ± 40 and 668 ± 62 sec for cardioprotector-1 and cardioprotector-2, respectively, thus exceeding this indicator by 2.5 and 3.3 times for the optimal composition (205 ± 13 sec, table 4).

The results of determining the activity of MV-CPK in rat plasma in vivo with the introduction of cardioprotectors with a changed composition of the components are given in table 7. It is seen that the use of cardioprotector-1 and cardioprotector-2 did not reduce the activity of the enzyme at the end of reperfusion compared with the control. In these cases, the activity of this marker of damage to cell membranes was on average 1.6 times higher than in experiments with the introduction of a cardioprotector of optimal composition. These data indicated that there was no reduction in sarcolemma ruptures of postischemic cardiomyocytes, which was in good agreement with an increase in the size of MI under the influence of cardioprotectors with an altered composition of components (Table 6).

Table 7 The effect of intravenous administration of cardioprotectors with a changed composition of components before PNA occlusion on the activity of MV-CPK in rat plasma in vivo The initial state End of reperfusion Control (n = 10) 430.2 ± 26.0 1868.0 ± 64.3 Cardioprotector of optimal composition (n = 10) 428.3 ± 20.1 1130.1 ± 47.2 ^a Cardioprotector-1 (n = 8) 429.0 ± 34.8 1704.8 ± 59.0 ^b Cardioprotector-2 (n = 8) 425.2 ± 24.5 1932.8 ± 64.4 ^b The data are presented as M ± m for a series of 8-10 experiments and are expressed in IU / L. Significantly different (p <0.05) from: control ^a : cardioprotector ^b .

The results obtained indicate that varying the content of components does not increase the protection of the myocardium from ischemic and reperfusion injury. On the contrary, these changes cause the cancellation of the protective effect of the drug for all investigated indicators. Thus, the composition of the cardioprotector indicated on page 4 is optimal. Its use provides a greater restriction on the size of myocardial infarction, better restoration of the metabolic state of the heart during reperfusion, greater stability of the membranes of postischemic cardiomyocytes, and less hemodynamic disturbances compared to traditionally used drugs.

Information sources

Claims (1)

A water-soluble composition with cardioprotective properties for intravenous administration, including d-glucose, potassium chloride, human genetically engineered insulin and water, characterized in that it additionally contains 1-aspartic acid potassium salt, 1-aspartic acid semi-magnesium salt and dinitrosyl complex iron (II) with glutathione in the following ratio of ingredients, wt.%:
d-glucose 23.40-28.60 potassium chloride 0.14-0.16 potassium salt of 1-aspartic acid 0.81-0.99 semi-magnesium salt of 1-aspartic acid 0.72-0.88 genetically engineered human insulin (in IU / l) 54.00-66.00 dinitrosyl complex of iron (II) with glutathione 0.19-0.23 water for injections rest,
while the pH of the solution is 7.4 ± 0.1 at 22 ° C.

Images