EA039287B1 - Method for the treatment of ischemic heart disease - Google Patents

Abstract

The invention relates to medicine, namely to means for the treatment of ischemic heart disease and its consequences. A method for the treatment of ischemic heart disease according to the invention comprises administering a pharmaceutical composition containing arginine hydrochloride, levocarnitine and water for injections. The technical result consists in broadening the range and assortment of pharmaceutical preparations for the treatment of ischemic heart disease and its consequences, creating a pharmaceutical preparation that is more effective due to synergism effect of active components.

Description

Technical field

The technical solution relates to the field of medicine, namely to agents for the treatment of coronary heart disease and its consequences.

Prior Art

The term coronary heart disease (abbreviated as IHD) is used to designate a group of cardiovascular diseases, which are based on myocardial damage, which is caused by disorders of the coronary circulation and occurs as a result of an imbalance between the delivery and metabolic oxygen demand of the heart muscle.

Myocardial oxygen demand is primarily determined by heart rate, myocardial contractility, heart size, and blood pressure. An increase in any of these indicators increases myocardial oxygen demand. Under normal conditions, there is a sufficient reserve of dilatation of the coronary arteries, providing, if necessary, a five-fold increase in coronary blood flow. Restriction of blood supply to the myocardium occurs due to a decrease in the lumen of the coronary artery by more than 50%. The discrepancy between the coronary blood flow and the metabolic needs of the heart muscle is always accompanied by myocardial ischemia, clinically manifested by an attack of angina pectoris, severe disorders of the heart rhythm and conduction, in some cases by the occurrence of myocardial infarction, and sometimes sudden death occurs.

According to Order No. 54 of the Ministry of Health of Ukraine in 2001, the following forms of coronary artery disease are provided.

1. Sudden coronary death.

Sudden clinical coronary death with successful resuscitation.

Sudden coronary death (lethal outcome).

2. Angina.

Stable exertional angina with functional class (FC).

Stable exertional angina, angiographically intact vessels (coronary syndrome X).

Vasospastic angina (angiospastic, spontaneous, variant, Prinzmetal).

3. Unstable angina.

Primary angina.

Progressive angina.

Early post-infarction angina pectoris (from 3 to 28 days of myocardial infarction).

4. Acute myocardial infarction.

Acute myocardial infarction with the presence of a pathological Q wave.

Acute myocardial infarction without pathological Q wave.

Acute myocardial infarction (unspecified).

Recurrent myocardial infarction (from 3 to 28 days).

Repeated myocardial infarction (after 28 days).

Acute coronary insufficiency.

5. Complications of myocardial infarction (indicating the time of occurrence).

Acute heart failure (T. Killip classes I-IV).

Violation of the heart rhythm and conduction.

Rupture of the heart is external (with hemopericardium, without hemopericardium) and internal (ventricular septal defect, atrial septal defect, chorda tendon rupture, papillary muscle rupture).

Thromboembolism of various localization.

Acute aneurysm of the heart.

Dressler Syndrome.

Postinfarction angina pectoris (after 3 to 28 days).

6. Cardiosclerosis.

Focal cardiosclerosis, namely:

post-infarction cardiosclerosis (indicating myocardial infarction, its location and time of development);

chronic aneurysm of the heart;

focal cardiosclerosis (without indication of myocardial infarction).

Diffuse cardiosclerosis.

7. Painless form of coronary artery disease.

The main etiological factor of coronary artery disease is atherosclerosis of the coronary arteries. Important factors contributing to its development are hyperlipidemia, arterial hypertension, high-calorie nutrition, obesity, diabetes mellitus, smoking, physical inactivity, genetic predisposition, age, male gender. Myocardial ischemia, which is associated with damage to the coronary arteries of a different origin (rheumatism, septic endocarditis, etc.), as well as hemodynamic disorders of non-coronary origin (aortic heart disease), does not belong to IHD and is considered

- 1 039287 as a secondary syndrome within the framework of nosological forms.

Coronary atherosclerosis is manifested in 95% of patients with coronary artery disease. Enlarging atherosclerotic plaque, hemorrhage at the base of the plaque with its disintegration, thrombus formation lead to a narrowing of the lumen or a complete obstruction of the patency, resulting in an organic obstruction of the coronary artery.

Myocardial endothelium also produces vasodilators: prostaglandin, prostacyclin and endothelial relaxing factor (abbreviated ERF), nitric oxide (NO), which are also antiaggregants. In IHD patients, the dynamic balance between endothelial vasodilating and antiplatelet factors, on the one hand, and vasoconstrictor and proaggregant factors, on the other hand, is disturbed. The latter begin to predominate, which leads to the development of coronary spasm and increased platelet aggregation. In the progression of coronary artery disease, an important role is played by disturbances in the hemostasis system, such as changes in platelet function, increased blood viscosity, inhibition of fibrinolysis - which can cause the development of intravascular thrombosis. The insufficiently developed network of collateral coronary blood supply matters. It has been proven that hyperproduction of catecholamines, which occurs in the case of stressful situations, can be the cause of myocardial damage. Attention should be paid to the consequences of functional physical overload of the heart.

One of the main drugs for the treatment of patients with coronary artery disease is arginine. Arginine (δ-guanidine-a-aminovaleric acid) is the main α-amino acid, the L-form of which is a semi-essential amino acid.

In smooth muscle cells of vessels, including coronary arteries, arginine interacts with SH-groups (nitrate receptors), forming nitric oxide (NO), which is similar in structure and action to ERF. Arginine, due to its properties, dilates arterioles and peripheral veins, reduces total peripheral vascular resistance, reduces venous outflow, and also dilates pulmonary vessels, helps reduce resistance in the pulmonary circulation and leads to regression of symptoms in pulmonary edema, reduces end-diastolic pressure and ventricular volume, thereby reducing myocardial oxygen demand. Also, arginine dilates the coronary arteries and prevents their spasm, reduces the diastolic tension of the ventricular wall, resulting in improved coronary blood flow in the ischemic zone.

The prior art method for the complex therapy of coronary heart disease is the drug Tivortin (web page at http://mozdocs.kiev.ua/likiview.php?id=23530), which contains arginine in the form of arginine hydrochloride and water for injection .

It is known from open sources that substances of the so-called class of metabolic correctors are widely used in cardiometabolic therapy - inhibitors of the oxidation of free fatty acids that affect the activity of enzymes involved in biochemical reactions.

A well-known representative of these inhibitors is levocarnitine (another name is L-carnitine). Levocarnitine facilitates the entry of long-chain fatty acids into the mitochondria of cells and thus provides a substrate for oxidation and energy generation, which significantly improves the recovery process of heart muscle cells in myocardial infarction. Levocarnitine inhibits the formation of atherosclerotic plaques in the blood vessels and promotes the resorption of plaques that have already formed.

Thus, due to its properties, levocarnitine reduces the contribution of such factors as hyperlipidemia, high-calorie nutrition, obesity, diabetes mellitus, smoking, physical inactivity, and age to the development of coronary artery disease.

From the prior art, pharmaceutical compositions for the treatment of coronary artery disease containing both arginine and levocarnitine are unknown. The publications note the use of a drug containing arginine and a drug containing levocarnitine in the treatment of coronary artery disease, but these drugs are not used together. Reception is carried out with a time interval.

Disclosure of invention

The technical challenge is to create a more effective way to treat coronary heart disease and its consequences.

The technical result is the expansion of the arsenal and range of pharmaceutical preparations for the treatment of coronary heart disease and its consequences, the creation of a pharmaceutical preparation that is more effective due to the synergy of the action of active components.

The problem is solved by a method for the treatment of coronary heart disease, which includes the introduction of a pharmaceutical composition that contains arginine hydrochloride, levocarnitine and water for injection, in the following ratio of components, in mg/ml:

arginine hydrochloride 37-47 levocarnitine 16-24 water for injection up to 1 ml

Advantageously, the method according to the technical solution may include the introduction of a pharmaceutical composition that contains arginine hydrochloride, levocarnitine and water for injection in the following ratio of components, in mg/ml:

- 2 039287 arginine hydrochloride 42 levocarnitine 20 water for injection up to 1 ml

Mainly, in the method according to the technical solution, coronary heart disease includes angina pectoris, exertional angina pectoris, spontaneous angina pectoris, myocardial infarction, postinfarction cardiosclerosis, cardiac arrhythmias, heart failure.

Arginine is used in pharmaceuticals in the form of a salt such as arginine hydrochloride. Further in the text, the term arginine will be understood as such a form of arginine as arginine hydrochloride.

Variant (variants) of the invention

The claimed pharmaceutical composition is a clear solution, the dosage form for use is a solution for infusion. The method of manufacturing the claimed pharmaceutical composition is shown below in examples 1-16.

Example 1

90 liters of water for injection are poured into a stainless steel reactor. The water is saturated with nitrogen until the residual amount of oxygen in the water is not more than 300 ppm. Then, 1 kg of arginine hydrochloride is charged into the reactor and stirred for a certain time until a clear solution is obtained. Then, 0.5 kg of levocarnitine is loaded into the reactor and stirred for a certain time until a clear solution is obtained. Water for injection is then added to the reactor, bringing the volume of the solution to 100 liters. The resulting solution is saturated with nitrogen to a residual amount of oxygen of not more than 300 ppm, after which the solution is filtered through two successive membrane filters. After filtration, the solution is poured into glass or polymer containers (bottles).

Example 2

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 1 kg and levocarnitine in the amount of 1.5 kg.

Example 3

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 1 kg and levocarnitine in the amount of 3 kg.

Example 4

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 1 kg and levocarnitine in the amount of 5 kg.

Example 5

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 3 kg and levocarnitine in the amount of 0.5 kg.

Example 6

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 3 kg and levocarnitine in the amount of 1.5 kg.

Example 7

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 3 kg and levocarnitine in the amount of 3 kg.

Example 8

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 3 kg and levocarnitine in the amount of 5 kg.

Example 9

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 6 kg and levocarnitine in the amount of 0.5 kg.

Example 10

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 6 kg and levocarnitine in the amount of 1.5 kg.

Example 11.

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 6 kg and levocarnitine in the amount of 3 kg.

Example 12.

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 6 kg and levocarnitine in the amount of 5 kg.

Example 13

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 10 kg and levocarnitine in the amount of 0.5 kg.

Example 14

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 10 kg and levocarnitine in the amount of 1.5 kg.

- 3 039287

Example 15

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 10 kg and levocarnitine in the amount of 3 kg.

Example 16

The claimed pharmaceutical composition is prepared similarly to the method described in example 1, while loading arginine hydrochloride in the amount of 10 kg and levocarnitine in the amount of 5 kg.

The manufacture of the claimed pharmaceutical composition is also possible in other ways.

To study the pharmaceutical effect of the application of the claimed pharmaceutical composition, a number of studies were conducted.

At the first stage, preclinical studies were carried out in order to study the specific activity of the claimed pharmaceutical composition.

Preclinical studies were carried out on rats with a model of pituitrin-isadrin myocardial infarction (MI for short), which were administered the claimed pharmaceutical composition, as well as other drugs - comparators.

In the process of conducting preclinical studies, the following indicators of the state of the animal organism were determined:

the level of creatine phosphokinase (abbreviated as CPK);

the level of myocardial creatine kinase isoenzyme (abbreviated as MB-CPK);

levels of adenosine triphosphate (ATP for short);

malate level;

lactate level;

catalase level;

the level of glutathione peroxidase (GPO for short);

the level of aldehyde phenylhydrazones (APH for short);

the level of carboxyphenylhydrazones (abbreviated as CFG);

morphofunctional characteristics of animal cardiomyocytes.

As morphofunctional characteristics of cardiomyocytes of animals, such indicators as the area of nuclei of cardiomyocytes were determined;

density of nuclei per 1 mm ² of myocardial area;

RNA concentration in the cytoplasm of cardiomyocytes;

density of nuclei of apoptotic and destructively altered cardiomyocytes per 1 mm ² of myocardial area.

To determine the effect of the content of the components of arginine and levocarnitine in the claimed pharmaceutical composition on the pharmaceutical effect, studies were conducted at various concentrations of arginine and levocarnitine in the claimed pharmaceutical composition. The concentrations of arginine and levocarnitine in the claimed pharmaceutical composition varied from 10 to 100 mg/ml for arginine and from 5 to 50 mg/ml for levocarnitine. The research results are given in table. one.

According to the results of the studies, it can be seen that the introduction of the claimed pharmaceutical composition with different contents of arginine and levocarnitine led to a decrease in the activity of CPK and MB-CPK in the blood. A significant decrease in CK and MB-CK in the blood is seen with an increase in the concentration of arginine / levocarnitine, respectively, to a level of 42/20 mg / ml. A further increase in the concentration of arginine and levocarnitine in the claimed pharmaceutical composition does not lead to a significant increase in the pharmaceutical effect.

Studies have been conducted to determine the indicators of energy metabolism in the myocardium of animals with MI. As a result, an increase in ATP levels and a decrease in lactate levels were observed.

Studies have also been conducted that show a decrease in oxidative stress markers aldehyde phenylhydrazones and carboxylphenylhydrazones, and an increase in the activity of antioxidant enzymes - catalase and glutathione peroxidase. The most pronounced pharmaceutical effect in terms of the above indicators is seen with an increase in the concentration of arginine / levocarnitine to the level of 42/20 mg / ml. A further increase in the concentration of arginine and levocarnitine in the claimed pharmaceutical composition does not lead to a significant increase in the pharmaceutical effect.

The results of histological studies showed a decrease in the change in the morphological and functional parameters of cardiomyocytes under the influence of ischemia. In the myocardium of the animals of the experimental group, a significant increase in the density of cardiomyocyte nuclei was recorded, which indicated a decrease in cell death and the preservation of a healthy myocardial structure. The most significant results are seen with an increase in the concentration of arginine / levocarnitine to the level of 42/20 mg / ml. A further increase in the concentration of arginine and levocarnitine in the claimed pharmaceutical composition does not lead to a significant increase in the pharmaceutical effect.

- 4 039287

Table 1

The influence of the concentrations of arginine and levocarnitine in the claimed pharmaceutical composition on the indicators of the state of the organism of animals

Indicators The concentration of arginine / levocarnitine, in mg / ml 10/5 20/10 30/12 37/16 42/20 47/24 50/27 70/35 100/50 CPK, IU/l 460.5± 20.1 457.3± 19.1 452.0± 22.0 444.3± 20.5 432.1± 23.1 430.3± 21.0 430.0±20 428.9± 18.0 427.5± 19 MW-CPK, IU/l 66.8± 5.5 59.3± 4.8 54.6±5 53.6± 4.7 41.1± 3.61 40.8± 4.7 40.1± 4.9 39.1± 5.0 38.1± 5.0 ATP, µmol/g 1.63± 0.11 1.65±0.12 1.67 ± 0.12 1.8± 0.15 2.10±0.17 2.11 ± 0.12 2.12 ± 0.10 2.14 ± 0.12 2.17 ± 0.10 Malate, µmol/g 0.19 ± 0.02 0.25±0.01 0.35±0.02 0.45+ 0.03 0.78±0.05 0.79±0.03 0.78±0.05 0.80±0.05 0.83 ± 0.04 Lactate, µmol/g 7.6± 0.52 6.5±0.3 5.9± 0.41 6.3 ± 0.3 5.15+ 0.21 5.14±0.20 5.13 ± 0.19 5.11 ± 0.20 5.08±0.22 Catalase, mkat/mg 10.1± 0.77 10.4±0.73 11.0 ± 0.9 11.5± 1.0 14.3± 1.12 14.5± 1.0 14.7± 1.2 14.9± 1.1 15.0± 1.2 GPO, µmol/mg/min. 67.9± 6.5 70.5± 7.1 76.4± 7.5 81.3± 8.1 93.7± 8.81 94.0± 8.2 94.1± 8.0 94.3± 8.3 95.0± 8.0 AFG, c.u./g 29.2± 2.3 25.7± 2.0 21.4+ 2.1 19.6± 1.8 18.1± 1.17 18.0± 1.8 17.8± 1.7 17.3± 1.6 16.5± 1.9 CFG, c.u./g 09.0+ 1.2 09.5± 1.6 11.2± 2.0 12.1± 1.8 14.31±0.87 14.33± 1.4 14.35± 1.6 14.38± 1.9 14.41± 1.9 Area of cardiomyocyte nuclei, µm ² 07.8± 0.2 08.3± 0.21 08.5± 0.23 09.3 ± 0.3 12.7 ± 0.30 12.75±0.3 12.78±0.4 12.81±0.3 12.82±0.35 Density of nuclei per 1 mm ² of myocardial area 6650± 177 6876 ± 182 7000+ 183 7076.4±176 7851.0 ±220.0 7851.5±150.5 7851.7±180.1 7852± 165 7852.2±172 The concentration of RNA in the cytoplasm of cardiomyocytes, EOP 0.050±0.002 0.055±0.002 0.057 ± 0.002 0.060±0.002 0.077 ± 0.002 0.078±0.002 0.079±0.001 0.081 ± 0.001 0.083±0.002 Density of nuclei of apoptotic and destructively altered cardiomyocytes per 1 mm ² of myocardial area 889.5± 11.0 700.1± 12.1 726.5± 11.3 735.3± 12.6 600.0± 13.0 559.8± 12.5 559.5± 12.0 559.1± 11.0 559.0± 12.3

To compare the pharmaceutical effects of arginine alone, levocarnitine alone, and arginine and levocarnitine together, a preclinical study was conducted in a rat pituitrin-isadrine myocardial infarction (MI) model.

To conduct these studies, the drug was used, which is the claimed pharmaceutical composition containing 42 mg/ml arginine and 20 mg/ml levocarnitine;

the first comparison drug containing arginine in the amount of 42 mg/ml;

the second reference drug containing levocarnitine in an amount of 20 mg/ml.

Animals were divided into the following groups:

intact animals (healthy animals);

MI (control) - these are animals with a model of pituitrin-isadrin myocardial infarction;

MI+arginine are animals with a model of pituitrin-isadrine myocardial infarction, which were injected with the first comparator drug;

MI + levocarnitine are animals with a model of pituitrin-isadrin myocardial infarction, which were injected with the second comparator drug;

MI+technical solution are animals with a model of pituitrin-isadrine myocardial infarction, which were injected with the claimed pharmaceutical composition.

As is known, an increase in the activity of creatine phosphokinase (CK) and myocardial isoenzyme CPK (CK-MB) indicate ischemic damage to the myocardium.

According to biochemical studies, it was found that the administration of arginine, levocarnitine and the claimed pharmaceutical composition to animals with MI led to a decrease in the activity of CK and CK-MB in the blood, and in the group of animals that received the claimed pharmaceutical composition, less pronounced hyperenzymemia was noted. The research results are given in

- 5 039287 tab. 2.

This indicates a pronounced cardioprotective effect of the claimed pharmaceutical composition, which exceeds the effect of arginine and levocarnitine.

table 2

The content of biochemical markers of ischemic myocardial damage in blood serum

Indicators Intact animals MI (Control) IM + arginine MI + levocarnitine IM + Technical solution CPK, IU/l 181.1+14.3 604.3±22.2 468.2±21.7* 580.5±19.0* 432.1±23.1* MW-CPK, IU/l 14.3±1.25 66.5±5.15 50.1±4.34* 60.1±3.6* 41.1±3.61*

* Changes are significant in relation to animals of the control group (p<0.05).

Also, the claimed pharmaceutical composition more pronounced improves energy metabolism in the myocardium of animals with MI. In animals receiving the claimed pharmaceutical composition, a significant increase in ATP levels was observed against the background of an increase in malate, which indicated the normalization of the Krebs cycle and a decrease in lactate levels, which demonstrated inhibition of glycolysis. The research results are given in table. 3.

Table 3

Indicators of energy metabolism in the cytosolic fraction of the heart of animals

Indicators Intact animals MI (Control) IM + arginine MI + levocarnitine IM + Technical solution ATP, µmol/g 2.91±0.18 1.62±0.12 1.71±0.16 1.75±0.14 2.10±0.17* Malate, µmol/g 0.67±0.02 0.19±0.02 0.21±0.07 0.39±0.05 0.78±0.05* Lactate, µmol/g 2.11±0.18 7.57±0.53 7.80±0.78 6.55±0.90 5.15±0.21*

* Changes are significant in relation to animals of the control group (p<0.05).

By the strength of the impact on NO/reduced thiols. The claimed pharmaceutical composition also turned out to be the leader.

Analysis of the obtained data given in table. 4 demonstrates the antioxidant effect of arginine, levocarnitine and the claimed pharmaceutical composition. A decrease in oxidative stress markers AFG and CPG is seen. Also, in the myocardium of animals treated with the claimed pharmaceutical composition, a significant increase in the activity of antioxidant enzymes - catalase and GPO was revealed, which gave the antioxidant effect of the claimed pharmaceutical composition a more pronounced character compared to the similar effect of the reference drugs.

Table 4

Indicators of the pro- and antioxidant system in the cytosolic fraction of the heart of animals

Indicators Intact animals MI (Control) IM + arginine MI + levocarnitine IM + Technical solution Catalase, mkat/mg 17.2±1.5 8.70±0.77 10.5±0.71 10.9±0.75 14.3±1.12* GPO, µmol/mg/min 154.2±11.2 73.7±6.83 80.4±7.55 81.8±7.80 93.7±8.81* AFG, c.u./g 10.2±1.21 35.7±2.11 29.7±2.16 32.1±2.00* 18.1±1.17* CFG, c.u./g 6.52±0.71 19.7±1.71 17.0+1.12 * 18.6±1.10* 14.31±0.87*

* Changes are significant in relation to animals of the control group (p<0.05).

Histological data also testify to the powerful cardioprotective effect of the technical solution. It was found that the administration of the claimed pharmaceutical composition to animals with MI leads to a decrease in the change in the morphological and functional parameters of cardiomyocytes under the influence of ischemia. In the myocardium of animals of the experimental group, a significant increase in the density of cardiomyocyte nuclei was recorded, which indicated a decrease in cell death and the preservation of a healthy structure of the myocardium - the results of the studies are shown in Table. 5.

- 6 039287

Table 5

Morphofunctional characteristics of animal cardiomyocytes

Indicators Intact animals MI (Control) IM + arginine MI + levocarnitine IM + Technical solution Area of cardiomyocyte nuclei, µm ² 15.1±0.40 11.2±0.27 11.7 ± 0.20 11.8±0.23 12.7±0.30* Density of nuclei per 1 mm ² of myocardial area 8877.0+ 320.0 6781.0± 177.0 7076.0± 211.0* 7246.0±209.0* 7851.0± 220.0* The concentration of RNA in the cytoplasm of cardiomyocytes, EOP 0.081 ± 0.002 0.062 ± 0.002 0.064± 0.002 0.066±0.002 0.077±0.002* Density of nuclei of apoptotic and destructively altered cardiomyocytes per 1 mm ² of myocardial area 133.0± 11.0 807.0+17.0 711.0± 11.0* 760.0±09.0* 600.0±13.0

* Changes are significant in relation to animals of the control group (p<0.05).

From the data in Table. 5 shows that the claimed pharmaceutical composition has the greatest cardioprotective effect, since it increases the RNA content in the cytoplasm of cardiomyocytes in parallel with a decrease in the death of cardiomyocytes, which indicates an increase in translation in the cell and activation of reparative processes.

From the foregoing, we can conclude that the claimed pharmaceutical composition based on arginine and levocarnitine has a pronounced cardioprotective effect due to the activation of the antioxidant system and optimization of the energy balance of cells. The pronounced cardioprotective effect of the claimed pharmaceutical composition is manifested in a decrease in the biochemical parameters of acute myocardial ischemia and the preservation of a healthy histological structure of the heart tissues of animals with a MI model.

Data analysis in table. 2-5 shows that the claimed pharmaceutical composition, compared to preparations containing only arginine and levocarnitine separately, has a more pronounced protective effect and has an unexpected technical result, namely, as a result of the analysis of the data obtained during the studies, we can talk about the manifestation of the claimed pharmaceutical composition of an unexpected synergistic effect.

In pharmacology, a special case of synergy, in which the effect of the simultaneous use of two or more active substances exceeds the total effect of the use of each of these substances separately, is called potentiation. As will be shown below by calculations, the use of components such as arginine and levocarnitine in the claimed pharmaceutical composition together gives a potentiation effect. Accordingly, the pharmaceutical composition according to the technical solution exhibits an unpredictable synergistic effect.

The calculation of the pharmaceutical effect for each of the three drugs and the determination of whether there is a potentiation effect was carried out according to the following method. First, the maximum possible pharmaceutical effect was calculated, which is the difference between the values of one of the indicators of the state of the organism in animals of the intact animals group and the corresponding indicator of animals of the MI group (control). The pharmaceutical effect for the first reference drug was determined as the difference between the values of one of the indicators of the state of the body in animals of the IM group (control) and the corresponding indicator of animals in the IM + arginine group and was expressed as a percentage relative to the value of the maximum possible pharmaceutical effect. The pharmaceutical effect for the second reference drug was determined as the difference between the values of one of the indicators of the state of the body in animals of the MI group (control) and the corresponding indicator of animals in the MI + levocarnitine group and was expressed as a percentage relative to the value of the maximum possible pharmaceutical effect. The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, was determined as the difference between the values of one of the indicators of the state of the body in animals of the MI group (control) and the corresponding indicator of the animals of the MI + technical solution group and was expressed as a percentage relative to the value of the maximum possible pharmaceutical effect. After that, the expected total pharmaceutical effect from the use of the first comparator drug and the second comparator drug together was calculated by summing the pharmaceutical effects of the first comparator drug and the second drug. Then the difference between the pharmaceutical effect of the drug, which is the claimed pharmaceutical composition, and the calculated expected total pharmaceutical effect was determined.

- 7 039287 the effect of using together the first comparator drug and the second comparator drug. If the pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, exceeds the calculated expected total pharmaceutical effect from the use of the first comparator drug and the second comparator drug together, it can be judged that there is a potentiation of the action of arginine and levocarnitine.

For example, according to biochemical studies, the use of all three drugs led to a decrease in the activity of CPK in the blood. The maximum possible pharmaceutical effect is 423.2. The pharmaceutical effect for the arginine preparation is 136.1, which is 32.16% of 423.2, the pharmaceutical effect for the levocarnitine preparation is 23.8, which is 5.6% of 423.2. Accordingly, the expected total pharmaceutical effect from the use of two reference drugs is 32.16+5.6=37.76%. The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, is 172.2, which is 40.69% of 423.2. The value of the pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, exceeds the value of the expected total pharmaceutical effect from the use of two reference drugs by 40.69-37.76=2.93%.

Calculations of the pharmaceutical effect of all three drugs were carried out in relation to each specific indicator of the state of the animal organism.

With the use of each drug, a decrease in the activity of MB-CPK in the blood was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 31.42%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 12.26%. The expected total pharmaceutical effect from the use of two reference drugs is 31.42+12.26=43.68%. The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, is 48.66%, which exceeds the expected total pharmaceutical effect from the use of two reference drugs in reducing the activity of MB-CPK in the blood by 48.6643.68 = 4.98%.

With the use of each drug, an increase in the level of ATP in the blood was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 6.98%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 10.08%. The expected total pharmaceutical effect from the use of two reference drugs is 6.98+10.08=17.05% The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, is 37.21%, which exceeds the expected total pharmaceutical effect from the use of two reference drugs to increase ATP rates by 37.21-17.05=20.16%.

With the use of each drug, an increase in the level of malate in the blood was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 4.17%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 41.67%. The expected total pharmaceutical effect from the use of two reference drugs is 4.17+41.67=45.83%. The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, is approximately 100%, which exceeds the expected total pharmaceutical effect from the use of two reference drugs in terms of increasing malate levels by 10045.83=54.17%.

With the use of each drug, a decrease in the level of lactate in the blood was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 14.10%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 18.68%. The expected total pharmaceutical effect from the use of two reference drugs is 14.10+18.68=32.78%. The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, is 44.32%, which exceeds the expected total pharmaceutical effect from the use of two reference drugs in terms of reducing lactate levels by 44.3232.78=11.54%.

With the use of each drug, an increase in the activity of catalase in the blood was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 21.18%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 25.88%. The expected total pharmaceutical effect from the use of two reference drugs is 21.18+25.88=47.06%. The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, is 65.88%, which exceeds the expected total pharmaceutical effect from the use of two reference drugs in terms of increasing the activity of catalase in the blood by 65.88-47.06 = 18.82%.

With the use of each drug, an increase in the activity of GPO in the blood was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 8.32%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 10.06%. The expected total pharmaceutical effect from the use of two reference drugs is 8.32+10.06=18.39%. The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, is 24.84%, which exceeds the expected total pharmaceutical

- 8 039287 the effect of the use of two comparator drugs on the increase in the indicators of GPO activity in the blood by 24.84-18.39=6.46%.

With the use of each drug, a decrease in the level of AFG in the blood was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 23.53%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 14.12%. The expected total pharmaceutical effect from the use of two reference drugs is 23.53+14.12=37.65%. The pharmaceutical effect for the drug is the claimed pharmaceutical composition and is 69.02%, which exceeds the expected total pharmaceutical effect from the use of two comparator drugs in reducing AFH in the blood by 69.02-37.65=3137%.

With the use of each drug, a decrease in the level of CFG in the blood was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 20.49%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 8.35%. The expected total pharmaceutical effect from the use of two reference drugs is 20.49+8.35=28.83%. The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, is 40.90%, which exceeds the expected total pharmaceutical effect from the use of two comparison drugs in terms of reducing blood CFG by 40.9028.83=12.06%.

With the use of each drug, an increase in the density of cardiomyocyte nuclei was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 12.82%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 15.38%. The expected total pharmaceutical effect from the use of two reference drugs is 12.82+15.38=28.21%. The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, is 48.66%, which exceeds the expected total pharmaceutical effect from the use of two comparison drugs in terms of increasing the area of cardiomyocyte nuclei by 38.46-28.21=10.26%.

With the use of each drug, an increase in the density of nuclei per 1 mm ² of myocardial area was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 14.07%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 22.19%. The expected total pharmaceutical effect from the use of two reference drugs is 14.07+22.19=36.26%. The pharmaceutical effect for the drug, which is a pharmaceutical composition according to the technical solution, is 51.05%, which exceeds the expected total pharmaceutical effect from the use of two comparison drugs in terms of increasing the density of nuclei per 1 mm ² of myocardial area by 51.05-36.26= 14.79%.

With the use of each drug, an increase in the content of RNA in the cytoplasm of cardiomyocytes was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 10.53%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 21.05%. The expected total pharmaceutical effect from the use of two reference drugs is 10.53+21.05=31.58%. The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, is 78.95%, which exceeds the expected total pharmaceutical effect from the use of two reference drugs in terms of increasing the RNA content in the cytoplasm of cardiomyocytes by 78.95-31.58=47.37%.

With the use of each drug, a decrease in the density of the nuclei of apoptotic and destructively altered cardiomyocytes was observed. The pharmaceutical effect expressed as a percentage for the arginine preparation is 14.24%, and the pharmaceutical effect expressed as a percentage for the levocarnitine preparation is 6.97%. The expected total pharmaceutical effect from the use of two reference drugs is 14.24+6.97=21.22%. The pharmaceutical effect for the drug, which is the claimed pharmaceutical composition, is 30.71%, which exceeds the expected total pharmaceutical effect from the use of two reference drugs in reducing the density of the nuclei of apoptotic and destructively altered cardiomyocytes by 30.71-21.22=9.50 %.

Thus, calculations for each indicator of the state of the animal organism show the effect of potentiating the simultaneous action of arginine and levocarnitine in the claimed pharmaceutical composition.

At the second stage, to study the effectiveness of the claimed pharmaceutical composition in the complex therapy of coronary heart disease, a randomized, open, comparative, parallel three-group clinical study was conducted on patients with acute coronary syndrome (ACS for short) without persistent ST segment elevations and unstable angina pectoris.

The study was conducted on 90 patients (30 in each group) of both sexes aged 25 to 75 years, in whom a clear picture of unstable angina pectoris was registered, or less than 24 hours have passed since the onset of symptoms of myocardial infarction.

Patients of the main group, in addition to the basic therapy, received the claimed pharmaceutical composition intravenously 1 time per day for 10 days. The patients of the first control group were prescribed basic therapy, which included nitroglycerin, aspirin and beta-blockers, as well as arginine (in individually prescribed doses). The patients of the second control group were prescribed basic therapy, which included nitroglycerin, aspirin and beta-blockers, as well as levocarnitine (in individually prescribed doses).

Evaluation of the effectiveness of treatment was carried out on the basis of the following criteria:

absence of clinical and ECG parameters of signs of myocardial ischemia;

stabilization of the state (lack of myocardial infarction, sudden death).

Additional criteria for the effectiveness of the treatment of ACS were elevated levels of troponin T, CK, or MB-CK;

instability of hemodynamics during the observation period from the moment of admission to the hospital; reduced contractile function of the left ventricle (less than 40%).

According to the results, during the period of inpatient treatment, patients of all three groups showed positive clinical dynamics, namely, the disappearance of angina attacks, a decrease in blood pressure, and an increase in exercise tolerance. Already on the third day after the administration of the claimed pharmaceutical composition, relapses of anginal pain occurred less frequently (20.8% of cases in the main group and 32.0% in the control groups). At the same time, there was a decrease in the need for the use of narcotic analgesics to treat recurrent pain syndrome (22.1% in the main group and 36.2 and 37%, respectively, in the second and third control groups). In addition, on the 3rd day of acute MI in patients of the main group, a decrease in the number of cases of atrioventricular blockades was recorded (4.2% in the main group and 12.6 and 11.8% in the second and third control groups, respectively). When analyzing the entire hospital period, it was found that in patients receiving the claimed pharmaceutical composition, almost 3 times less often than in the control groups, all cases of atrioventricular blockade were observed. The frequency of registration of ventricular extrasystoles decreased on days 7 (by 36.2%) and on days 10 (by 44.6%) of the disease.

To detect necrosis in the myocardium, the level of cardiac troponins was determined, as well as the mass of the MB fraction of CPK. Peak CPK in patients of the main group (declared pharmaceutical composition) was 3.16 ± 0.09 mkat / l, in patients of the control groups - 3.21 ± 0.12 and

3.33±0.10 µkat/l; MV-CPK - 0.24±0.02, in patients of the control groups - 0.25±0.01 and 0.27±0.02 mkat/l, which indicates insignificant differences in the volume of initially necrotic myocardium. The introduction of the claimed pharmaceutical composition in the first hours on the background of traditional therapy reduced the time to reach the peak of CPK activity in patients of the main group and amounted to 13.5 ± 0.6 hours, in patients of the control groups this figure reached 17.1 ± 0.8 and 17.5 ±0.5 h respectively; MV-CPK - 9.9±0.4, 13.9±0.6 and 13.6±0.4 hours, respectively.

At the same time, the area of necrotic lesion (calculated from the change in the activity of MB-CPK in the blood serum) was 26.4% less in patients of the main group compared to the controls: 45.4±2.1, 61.6±2.9 and 61 .3±2.0 g/eq. respectively. This was due to a reduction in the time of normalization of the activity of CPK and MB-CPK in the blood serum by an average of 8.1 hours (main group) and 9.4 and 9.2 hours (control groups), respectively. In comparison, the early onset of the peak of enzymes (CPK, MVKFK) indicates a more rapid formation of the necrosis zone, and a reduction in the timing of their washout indicates the prevention of further damage to healthy cardiomyocytes in patients of the main group (see Table 6).

Table 6

Comparison results using paired Student's t-test of biomarkers for each group of patients

Parameter Group df t statistics p value Statistically significant differences* MV-KFK Main 46 190.439 0.000 Significant Control No. 1 47 169.518 0.000 Significant Control №2 48 170,000 0.000 Significant KFK Main 46 123.492 0.000 Significant Control No. 1 47 105.987 0.000 Significant Control №2 48 105,900 0.000 Significant Troponin Main 46 19.065 0.000 Significant Control No. 1 47 4.494 0.000 Significant Control №2 48 5,000 0.000 Significant

* The conclusion was made at a significance level of 0.05;

tcrit=2.012 at f=46, tcrit=2.011 at f=47.

The study also noted a number of positive general clinical changes. In the main observation group, the pain syndrome regressed faster than in the control group. So, in the first 12 hours after the development of intense pain syndrome, there was no significant difference in its severity between the groups. On the third day of treatment compared with day 1 in the three groups, the intensity of the pain syndrome decreased significantly. From the 7th day of therapy, the degree of pain syndrome decreased more pronounced in the main group compared to the control group. Until the end of the observation period, this trend continued. The decrease in the intensity and frequency of anginal pain was accompanied by a decrease in the need for nitrates in patients of the main group.

Surveys of patients during the study showed that the introduction of the claimed pharmaceutical composition, starting from the first days of the development of ACS, has a positive effect on the subjective complaints of patients. Compared with the control groups, patients of the main group up to 10 days showed a significant decrease in such subjective sensations as a sense of fear, internal anxiety, weakness, pain in the heart or other parts of the chest, and a feeling of lack of air. Thus, the use of the claimed pharmaceutical composition against the background of traditional therapy for ACS without ST segment elevation had a positive effect on the main clinical parameters.

As for the echocardiographic parameters, they were recorded before and 10 days after admission to the hospital. According to the echocardiogram (abbreviated EchoCG), all three groups were statistically the same at the beginning of the study.

There were no significant changes in the size of the left ventricular cavity (end diastolic volume (abbreviated as EDV) of the LV) in the three groups in the follow-up dynamics. At the same time, in the main group, in contrast to the control group, there was a significant increase in the ejection fraction (EF) of the LV after 7 days of treatment. When evaluating the results of a dynamic study of EchoCG in the main group against the background of standard therapy, it was revealed that the early administration of the claimed pharmaceutical composition has a positive effect on cardiohemodynamics, reducing the dilatation of the LV cavity, as a result of which a decrease in the end-systolic volume (abbreviated ESV) of the LV was revealed by the 10th day of observation. At the same time, in the control groups, there was a trend towards an increase in the end-diastolic index on days 7 and 10 of acute MI. The LV ejection fraction increased in patients of all groups, but its increase on the tenth day of acute MI was more significant in the main group: 9.3, 6.1 and 5.7% in the control groups, respectively. The obtained EchoCG data allow us to conclude that the use of the claimed pharmaceutical composition as part of the basic standard therapy significantly and effectively affects LV remodeling in ACS without ST segment elevation.

The assessment of the overall effectiveness of the drugs was carried out using an integral variable, which included the stabilization of the patient's condition, the presence of complications, cases of recurrent myocardial infarction and sudden death.

In patients of the main group, one Q-forming MI and one MI without a Q wave were registered in the control groups. The reasons for the development of MI did not directly depend on the treatment and were caused by the duration and intensity of the pain syndrome before the start of therapy, as well as non-compliance by patients with the prescribed restriction of the motor regimen. In both cases, MI proceeded without complications and developed within the first 12 hours from the moment of admission to the hospital. In the total number of observations (90), one case of MI is 3.3% in relation to 30 patients of the main group, two cases of MI is 6.67% in relation to 30 patients of each of the control groups. There were no deaths from ACS in one of the groups. The results of the analysis of this variable by the methods of descriptive statistics (frequency and percentage in%) are given in Table. 7.

Based on the results, it was concluded that the treatment efficacy of the groups was statistically significantly different.

Thus, the use of the claimed pharmaceutical composition in the main group of patients, in addition to significantly earlier stabilization of ACS symptoms, made it possible to achieve a significant reduction in early post-infarction angina pectoris and ventricular tachyarrhythmia (ventricular fibrillation/ventricular tachycardia), which are life threatening.

The conclusion about the increase in the effectiveness of treatment using the claimed pharmaceutical composition was made taking into account the confidence intervals. The calculation results are given in Table. eight.

Thus, due to the fact that the lower limit of the 95% confidence interval for the difference in proportions is greater than the limits of the effective zone (0%), it can be concluded that complex therapy using the claimed pharmaceutical composition is more effective than basic therapy in control groups.

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Table 7

Evaluation of the effectiveness of treatment with investigational and reference drugs

Efficiency mark Main group n=30 Control group №1 n=30 Control group №2 n=30 η % η % η % Postinfarction angina one 3.3 2 6.67 one 3.33 MI recurrence one 3.3 2 6.67 2 6.67 Atrial fibrillation one 3.3 2 6.67 2 6.67 Ventricular fibrillation/ventricular tachycardia one 3.3 3 10.0 four 13.33 The drug is effective 26 86.67 21 63.33 21 63.33 The drug is not effective four 13.33 9 36.67 9 36.67

Table 8

The results of the analysis of the effectiveness of treatment

statistic Meaning Type I error probability (a) 0.025 Percentage point of the standard normal distribution for a 1.96 The boundary of the zone of exceeding efficiency (%) 0.0 Proportion of positive results for the main group (%) 87.2 Main group size 47 Proportion of positive results for the control group (%) 66.7 Control group size 48 Share difference (%) 20.5 standard error 8.37 Lower limit 95% CI 4.1 Upper limit 95% CI 36.9

Summing up the data of preclinical and clinical studies, we can conclude that the claimed pharmaceutical composition based on 2 active ingredients - arginine and levocarnitine - increases the effectiveness of complex therapy for coronary heart disease, improves the prevention and treatment of the consequences of coronary artery disease, improves the quality of life of patients by reducing the frequency of attacks angina pectoris and improved survival rates.

From the above research results, it can be seen that

1) the pharmaceutical composition according to the claimed technical solution has a significantly greater cardioprotective effect in MI when compared with preparations containing separately arginine and levocarnitine, and is ahead of the reference drugs in terms of activity and shows an unexpected effect of potentiating the action of arginine and levocarnitine;

2) the pharmaceutical composition according to the claimed technical solution has a greater anti-ischemic effect when compared with preparations containing separately arginine and levocarnitine, and is ahead of the comparison drugs in terms of activity and shows an unexpected effect of potentiating the action of arginine and levocarnitine;

3) the pharmaceutical composition according to the claimed technical solution has a greater antioxidant effect when compared with preparations containing separately arginine and levocarnitine, and is ahead of the reference drugs in terms of activity and shows an unexpected effect of potentiating the action of arginine and levocarnitine.

The above indicates that the claimed pharmaceutical composition according to the technical solution is highly effective and promising for implementation in medical practice as a means for the treatment of coronary heart disease and its consequences.

The given examples of implementation of the technical solution only illustrate it and do not limit it in any way.

Claims (3)

1. A method for the treatment of coronary heart disease, which includes the introduction of a pharmaceutical composition that contains arginine hydrochloride and water for injection, characterized in that the pharmaceutical composition additionally contains levocarnitine in the following ratio of components, in mg/ml: arginine hydrochloride - 37-47; levocarnitine - 16-24; water for injection - up to 1 ml. 2. Method for the treatment of coronary heart disease according to claim 1, characterized in that the pharmaceutical composition contains arginine hydrochloride, levocarnitine and water for injection in the following ratio, in mg/ml: arginine hydrochloride - 42; levocarnitine - 20; water for injection - up to 1 ml. 3. A method for the treatment of coronary heart disease according to claims 1, 2, characterized in that coronary heart disease includes angina pectoris, exertional angina, spontaneous angina pectoris, myocardial infarction, postinfarction cardiosclerosis, cardiac arrhythmias, heart failure. O Eurasian Patent Organization, EAPO Russia, 109012, Moscow, Maly Cherkassky per., 2