RU2693825C1 - Inosine as an anthracycline toxicity corrector - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention refers to medicine, namely cardiology and oncology. Proposed is the use of inosine in concentration of not less than 50 mcmole/l in cardiac perfusion as an anthracycline toxicity corrector of doxorubicin by reducing diastolic stress by more active output of Ca²⁺ of cardiomyocytes.

EFFECT: inosine in the declared conditions completely eliminates toxic effect of doxorubicine on cardiac muscle, which consists in depression of contractile function and violation of Na⁺ dependent streams of Ca²⁺ (Na⁺-Sa²⁺ exchange) in an intact isolated heart.

1 cl, 1 dwg, 1 tbl

Description

The invention relates to medicine, namely cardiology and oncology.

Some drugs have a significant cardiodepressive effect. An undesirable side effect is attributed to their ability to disrupt the transport of Ca ^{2+ ions} , the most important component of the action potential of pacemakers. A significant role is played by sodium ions Na ⁺ , ensuring the regulation of the concentration of calcium ions in cardiac cells, denoted as Na ⁺ -Ca ²⁺ exchange. Some antibiotics and anthracycline cytostatics - Chlorpromazine, Polymyxin, Doxorubicin - have an inhibitory effect on this transport system.

Doxorubicin [the drug register https://www.rlsnet.ru/mnn_index_id_99.htm#xarakteristika--veshhestva-doksorubicin] is known to be a powerful anti-tumor antibiotic used to treat many types of cancer. A serious complication in the treatment with doxorubicin is its cardiotoxic effect. The incidence of various cardiac complications in patients taking doxorubicin reaches 36%.

The exact mechanism of the toxic action of doxorubicin on the heart is still unknown. There is evidence of the inhibitory effect of Doxorubicin on the process of Na ⁺ -Ca ²⁺ metabolism in isolated fragments (vesicles) of the myocardial sarcolemma [Carafoli E. Calcium transport sarcolemma vesicles. Naunyn - Schmiederberg's Arch / E. Carafoli. // Pharmacol. - 1982. - V. 321. - R. 7.]

The known effect of Doxorubicin inhibition of the process of Ca ²⁺ absorption in an isolated rat heart during the Na ⁺ -Ca ²⁺ exchange [Alabovsky VV, Vinokurov AA, Maslov OV The selectivity parameters of the Na-Ca ion-exchange system to changes in the extracellular concentration of sodium ions during hypoxia and ischemia of the heart, as well as under the influence of certain cardiotropic drugs // System analysis and management in biomedical systems. - 2010. - T. 9, N 1. - p. 138-141.]

Existing methods of reducing the toxic effects of chemotherapy on the heart in the treatment of cancer patients are expensive and ineffective. There is information about the use of N-acetylcysteine preparations for this purpose, phenylamines, coenzyme Q10, vasopressin antagonists [Patent A61P 9/04 by Liu Yunge (US), Cambayasi Juniti (US) (RU 2470643) and others]. The cost of these drugs is high, it was not possible to achieve a significant reduction in the cardiotoxicity of Doxorubicin.

In this regard, it is of great practical importance to search for drugs that can reduce the toxic effect of Doxorubicin on Na ⁺ -Ca ²⁺ metabolism in the heart.

Known drug Inosine [drug register https://www.rlsnet.ru/mnn_index_id_644.htm] - nucleoside, the precursor of ATP. Substrate stimulates the synthesis of adenine nucleotides, increases the activity of some enzymes of the Krebs cycle, has antiarrhythmic, antihypoxic, metabolic effects. According to the manufacturer's instructions, the use of the substance Inosine is known for the following: IHD (myocardial infarction, coronary insufficiency, cardiac arrhythmias), congenital and acquired heart defects, rheumatic heart defects, cardiomyopathy of various origins, coronary atherosclerosis, digitalis intoxication, myocarditis, “lung heart” loads, infectious and endocrine genesis), liver diseases (acute and chronic hepatitis, cirrhosis, alcoholic and medicinal injuries of the liver, fatty degeneration of the liver ), Gastric ulcer and duodenal ulcers, urokoproporfirii. Poisoning drugs, alcoholism, radiation leukopenia (prophylaxis), operations on an isolated kidney (as a means of pharmacological protection in case of temporary shutdown of the blood circulation of the operated organ).

In the available literature there is no information on the use of Inosine for reducing anthracycline toxicity.

The technical result is to eliminate the toxicity of anthracycline antibiotics, which is represented by doxorubicin, to reduce the manifestation of its side effects and the material costs of treatment.

When choosing the necessary drug for effective protection of the heart, we paid attention to experimental data, in which significant nucleotide and nucleoside damage to the heart was found to be significantly attenuated under the conditions of the so-called “calcium paradox”.

Therefore, the drug Inosine was taken as a prototype, with the intended protective effect.

The technical result is achieved by adding Inosine at a concentration of at least 50 μmol / l to the extracellular medium of the heart in order to eliminate the toxic effects of Doxorubicin. To assess the protective effect of Inosine, we studied the transport of Ca.²⁺conjugated with a change in the transmembrane gradient of Na⁺, as well as contractile activity in an isolated rat heart. Such a methodical approach made it possible to preserve not only the anatomical integrity of the myocardial tissue, but also the physiological and biochemical processes associated with the work of NA.⁺-Ca²⁺ exchange [Pat. 2400825 Russian Federation, IPC G09B 23/28 A61B 5/04. Method for simultaneous registration of Na⁺ - dependent flows Sa²⁺ and functional state of a whole isolated heart in an experiment / V.V. Alabovsky, A.A. Vinokurov, O.V. Maslov; applicant and patent holder VGMA them. N.N. Burdenko Roszdrava. - №2009122349/14; publ. 09/27/2010 // Bull. - №27. - 3 p.]

The experiments were carried out on laboratory animals in strict accordance with modern requirements of the rules of laboratory practice for experimental (preclinical) research in the Russian Federation (GOST 351000.3-96 and 51000.4-96)

The main object of study were white rats of the Wistar line. In total, 48 animals of both sexes weighing 200–250 g were used in the work. Each series of experiments was carried out on 7–10 rats, depending on the reproducibility of the research results.

Under ether anesthesia, the animal was decapitated. The heart was quickly removed and placed in a chilled perfusion solution. In the study of Na ⁺ -Ca ²⁺ metabolism, perfusion of the heart was carried out at a constant speed using a peristaltic pump under pressure control in the aorta, which ranged from 60-70 mm Hg. Art. The speed of the coronary duct ranged from 8 to 10 ml / min, depending on the mass of the ventricles of the heart.

In the aorta of an isolated heart, a solution of the following composition was supplied: NaCl - 140 mmol / l, NaHCO ₃ - 2 mmol / l, KCl - 15 mmol / l, Tris-OH - 5 mmol / l, (pH 7.4), CaCl ₂ - 0.7 mmol / l, glucose - 11 mmol / l. Thus, the total concentration of sodium ions [Na ⁺ ] in the solution was 142 mmol / l.

In order to eliminate the influence of the mechanical activity of the heart on the process of accumulation and release of Ca ²⁺ during Na ⁺ -Ca ²⁺ exchange, cardiac activity was stopped with the help of a high extracellular concentration of potassium - 15 mmol / l. Depolarization of cardiomyocytes completely blocked their electrical activity.

Experiments began after 15 minutes of heart adaptation and stabilization of metabolic processes and contractile function of the heart.

The initiation of sodium-dependent absorption of calcium ions (reverse direction of Na ⁺ -Ca ²⁺ exchange) with an isolated rat heart was performed by reducing the concentration of sodium chloride in the perfusion solution.

The hyponatric perfusion solution was composed of: NaCl - 10 mmol / l, NH ₄ Cl - 130 mmol / l, NaHCO ₃ - 2 mmol / l, KCl - 15 mmol / l, Tris-OH - 5 mmol / l, (pH 7.4), CaCl ₂ - 0.7 mmol / l, glucose -11 mmol / l. Thus, this solution differed from the initial low amount of sodium ions [Na ⁺ ] –12 mmol / l.

Perfusion of the hypo-sodium solution, depending on the purpose of the experiment, was carried out from 1 to 5 minutes. Then the perfusion was transferred to the original solution containing the physiological concentration of sodium. At the same time observed the emission of Ca ^{2 +} from the heart (straight direction Na ⁺ -Ca ^{2 +}  exchange).

The calcium concentration in the perfusion solution flowing from the heart was continuously measured during the entire test period. Using a peristaltic pump, the perfusion solution was mixed with a metal indicator - arsenazo-III.

Arsenazo-III (2,7-bis - [(2-arsonophenyl) azo] -1,8-dioxy 3,6 naphthalene-disulfonic acid) is a reagent for the photometric determination of calcium ions. The maximum light absorption by the arsenazo-III-Ca ²⁺ complex was recorded at a wavelength of 660 nm. The metal indicator has high selectivity and sensitivity to calcium ions. In scientific studies, it is used to measure the intracellular concentration of Ca ²⁺ , the fluctuations of which are extremely small and range from 10 ^-6 to 10 ^-8 mol / l.

The resulting colored reaction product between the Ca ²⁺ perfusion solution and the metal indicator was passed through a 1 ml quartz flow cell placed in the registration unit of the SF-46 spectrophotometer (OKB SPECTR CJSC, Russia). The readings of the spectrophotometer were continuously recorded using the external module of the analog-digital converter ZET 220 and the software ZETLAB BASE (Enterprise "Electronic technologies and metrological systems", Russia).

The calibration of the system was carried out by adding calcium chloride to perfusion solutions, increasing the calcium concentration by 20 µmol / L. Heart contractions were recorded in the isovolumic mode using a balloon inserted into the cavity of the left ventricle of the heart. The pressure developed was calculated as the difference in systolic and diastolic pressure.

When registering contractions of the heart, perfusion solutions with physiological concentration of NaCl (142 mmol / l) and hypo-sodium solutions (12 mmol / l) were used in the same composition, differing in the concentration of KCl, which was 5 mmol / l and CaCl ₂ in an amount of 2 mmol / l .

The abbreviations were recorded using an Bentley lab. Europe electromagnet, the signals from which were applied to an external ZET 220 analog-to-digital converter module having ZETLAB BASE software.

The concentration of Doxorubicin in the perfusion solution was calculated based on its amount injected into the blood during treatment. With a single injection of 10 mg of the drug per 5 liter of blood creates a concentration of approximately 4 µmol / L. In our experiments, we added the drug to perfusion solutions to the same concentration - 4 µmol / l.

According to the instructions for use of Inosine a, a single dose of the drug is at least 200 mg / l of blood (200 μmol / l). However, we used the minimum concentration of Inosine (four times less) - 50 µmol / L of the perfusion solution.

All obtained data were checked for the probability distribution, and in the case when the sample corresponded to the normal distribution, statistical data processing was performed using Student's t-test. When non-compliance with the criteria of the normal distribution, the non-parametric Mann-Whitney-Wilcoxon U-test was used to compare the samples. All calculations were performed using statistical analysis packages.

Experiments have shown that the differences in Na ⁺ -Ca ²⁺ metabolism in the control and experiments were associated with different rates of Ca ²⁺ accumulation by the heart muscle. From table 1, it can be seen that Doxorubicin has the property of significantly reducing the rate of Ca ²⁺ absorption by the heart.

The results of the experiment show a significant decrease in the amount of Ca ²⁺ absorbed during the period of its transport inside the cardiomyocytes and a decrease in the rate of increase in diastolic myocardial tension, which immediately, after a slight rise, monotonously decreases to the initial level of control (Figure 1, A). Accordingly, the amount of Ca ^{2+ released} from the heart, in the presence of Doxorubicin, was lower than in control experiments.

It can be seen that under the influence of Doxorubicin, the reaction to repeated attempts to activate the Na ⁺ -Ca ²⁺ exchange in the heart in the forward and reverse directions was completely absent. In contrast to the control experiments, switching the perfusion of the heart to the initial solution, with the physiological level of NaCl, as well as repeated attempts to activate the Na ⁺ -Ca ²⁺ exchange did not cause any responses from the left ventricular contractile apparatus (Figure 1, B). This indicates a significant violation of the Na ⁺ -Ca ²⁺ exchange mechanism.

Thus, the obtained results indicate that Doxorubicin, disrupting the mechanism of Ca ²⁺ transport in the heart, weakens its entry into cardiomyocytes under conditions of reversal of Na ⁺ -Ca ²⁺ metabolism and its release from cells in the forward direction of the Na ⁺ -Ca system ²⁺ exchange. As a result, the cardiodepressive effect of Doxorubicin on the heart is observed.

In another series of experiments, Doxorubicin (4 µmol / L) and Inosine at a concentration of 50 µmol / L were added simultaneously to the perfusion solutions. At the same time, the inhibitory effect of Doxorubicin on the rate of Ca ²⁺ absorption was significantly weakened under the influence of Inosine (Table 1). Thus, Inosine in combination with Doxorubicin restored the ability of the Na ⁺ -Ca ²⁺ metabolic system of the heart to accumulate Ca ²⁺ under the conditions of reversion of the Na ⁺ -Ca ²⁺ metabolism.

It is important to note that Inosine not only eliminated the inhibitory effect of Doxorubicin on the process of Ca ²⁺ absorption. His ability to more actively release an excess of Ca ²⁺ from the heart muscle was noted, compared not only with experiments with Doxorubicin but also with control experiments (Table 1).

Table 1 The effect of Doxorubicin and Doxorubicin with Inosine on the parameters of the Na-Ca exchange of an isolated rat heart. Control Doxorubicin Doxorubicin + Inosine (n = 8) (n = 8) (n = 9) Ca ²⁺ entry rate (μmol / min · g) 80.5 + 3.4 32 + 2.0 88.9 + 1.5 p * <0.001 p *> 0.5; p ** <0.001 The rate of release of CA ²⁺ (µmol / min · g) 42.4 + 2.7 35.7 + 3.0 68.1 + 2.6 p *> 0.1 p *> 0.5; p ** <0.02 The amount of absorbed Ca ²⁺ in 60 seconds (nmol / g) 52.4 + 1.9 21.9 + 1.8 54.9 + 2.3 p * <0.001 p *> 0.5; p ** <0.001 The number of derived CA ²⁺ in 60 seconds (nmol / g) 23.9 + 1.2 25.5 + 0.2 40.8 + 1.8 p *> 0.2 p * <0.01; p ** <0.02

Legend: n-number of animals in groups of experiments;

p * <0,001 - comparison is given with the control;

p ** <0,001 - comparison given with doxorubicin

The results of the study show that the ability to maximize Ca ^{2+ by the} myocardium under the influence of Inosine increased markedly.

This conclusion clearly demonstrates the protocols of registration of the contractile activity of the heart (Figure 1, B). First, there is a recovery in contractions in response to the onset of perfusion with a solution with the same physiological concentration of sodium. Secondly, the weakening of diastolic tension, which correlates at this moment with a more active release of calcium ions by means of Na ⁺ -Ca ²⁺ exchange, is clearly recorded.

Records of rat myocardial contractions using different combinations of substances indicate that the onset and continuation of perfusion with a solution containing a reduced concentration of sodium chloride (12 mmol / l); in experiments of the series “B”, a decrease in diastolic tension is observed. The combined use of Doxorubicin with Inosine results in a decrease in diastolic voltage due to a more active release of calcium ions from cardiomyocytes. The relaxation of calcium load on the myocardium initiates the restoration of heart contractions.

Thus, we proposed the use of Inosine as a drug that completely eliminates the toxic effect of Doxorubicin on the heart muscle. The effect of the drug on the myocardial contraction mechanism (Na ⁺ -Ca ²⁺ metabolism) has been proven. Inosine at a concentration of not less than 50 µmol / L eliminates the cardiotoxicity of anthracycline drugs, for example, Doxorubicin (4 µmol / L), which consists in depression of the contractile function of the heart and a violation of Na ⁺ dependent ^{Ca2 +} fluxes (Na ⁺ -Ca2 ⁺ metabolism) in intact isolated heart.

Description of the figures.

Figure. 1. Record the rat myocardium contractions in the control (A) and during the perfusion with Doxorubicin (B) and the mixture of Doxorubicin with Inosine (C)

1 - heart contractions in the initial state;

2 - the beginning and continuation of perfusion with a solution containing a reduced concentration of sodium chloride (12 mmol / l);

3 - switching perfusion of the heart to a perfusion solution containing the initial concentration of sodium chloride (142 mmol / l).

4 - repeated inclusions of a hypo-sodium solution

5 - repeated inclusions of a solution with a physiological content of sodium chloride do not cause reciprocal contractile reactions of the heart. The invention relates to medicine, namely cardiologists and oncology.

Some drugs have a significant cardiodepressive effect. An undesirable side effect is attributed to their ability to disrupt the transport of Ca ^{2+ ions} , the most important component of the action potential of pacemakers. A significant role is played by sodium ions Na ⁺ , ensuring the regulation of the concentration of calcium ions in cardiac cells, denoted as Na ⁺ -Ca ²⁺ exchange. Some antibiotics and anthracycline cytostatics - Chlorpromazine, Polymyxin, Doxorubicin - have an inhibitory effect on this transport system.

It is known that the drug Doxorubicin [drug register https://www.rlsnet.ru/mnn_index_id_99. htm # xarakteristika-veshhestva-doksorubicin] is a powerful antitumor antibiotic used to treat many types of cancer. A serious complication in the treatment with doxorubicin is its cardiotoxic effect. The incidence of various cardiac complications in patients taking doxorubicin reaches 36%.

The exact mechanism of the toxic action of doxorubicin on the heart is still unknown. There is information about the inhibitory effect of Doxorubicin on the process of Na ⁺ -Ca ²⁺ exchange in isolated fragments (vesicles) of myocardial sarcolemma [Carafoli E. Calcium transport systems of heart and sarcolemma vesicles. Naunyn-Schmiederberg's Arch / E. Carafoli. // Pharmacol. - 1982. - V. 321. - P. 7.]

Known effect of Doxorubicin inhibition process of absorption

Ca ²⁺ in an isolated rat heart during Na ⁺ –Ca ²⁺ metabolism [Alabovsky V.V., Vinokurov A.A., Maslov OV The selectivity parameters of the Na-Ca ion-exchange system to changes in the extracellular concentration of sodium ions during hypoxia and ischemia of the heart, as well as under the influence of certain cardiotropic drugs // System analysis and management in biomedical systems. - 2 010. - V. 9, N 1.- P.138-141.]

Existing methods of reducing the toxic effects of chemotherapy on the heart in the treatment of cancer patients are expensive and ineffective. There is information about the use of N-acetylcysteine preparations for this purpose, phenylamines, coenzyme Q10, vasopressin antagonists [Patent A61P 9/04 by Liu Yunge (US), Cambayasi Juniti (US) (RU 2470643) and others].

The cost of these drugs is high, it was not possible to achieve a significant reduction in the cardiotoxicity of Doxorubicin.

In this regard, it is of great practical importance to search for drugs that can reduce the toxic effect of Doxorubicin on Na ⁺ -Ca ²⁺ metabolism in the heart.

Known drug Inosine [drug register https://www.rlsnet.ru/mnnindexid644.htm] - nucleoside, the precursor of ATP. Substrate stimulates the synthesis of adenine nucleotides, increases the activity of some enzymes of the Krebs cycle, has antiarrhythmic, antihypoxic, metabolic effects. According to the manufacturer's instructions, the use of the substance Inosine is known for the following: IHD (myocardial infarction, coronary insufficiency, cardiac arrhythmias), congenital and acquired heart defects, rheumatic heart defects, cardiomyopathy of various origins, coronary atherosclerosis, digitalis intoxication, myocarditis, “lung heart”

Claims (1)

Use of Inosine in a concentration of at least 50 µmol / L when perfusing the heart as a corrector of anthracycline toxicity of Doxorubicin, by reducing diastolic voltage due to a more active release of CA ²⁺ from cardiomyocytes.

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