RU2741906C1 - Method of atherogenesis correction in experiment using 1-(germatran-1-yl)-1-oxyethylamine - Google Patents

Abstract

FIELD: biochemistry.SUBSTANCE: invention relates to a method for reducing the total (sum) activity of a lysosomal lipolytic enzyme, acid phospholipase A1. The method includes administering a drug, which is a composition containing 1-(germatran-1-yl)-1-hydroxyethylamine and a pharmaceutically acceptable aqueous carrier. The agent is administered to the animal intramuscularly at a dose of 5.0 mg of active substance / kg of animal weight daily for 2 months.EFFECT: expanded methods of influencing the activity of acidic phospholipase A1 by using the agent - 1- (germatran-1-yl)-1-hydroxyethylamine, due to its newly identified biological activity, namely the ability to reduce the total activity of acidic phospholipase A1.4 cl, 2 tbl, 1 ex

Description

The invention relates to medicine, pharmacology and biology and can be used to increase the resistance of the vascular system to cholesterol in the development of hyperlipidemia in the pathogenesis of atherosclerotic lesions.

It is known that at the early stage of atherogenesis, the endothelium is activated, which releases on its surface adhesion molecules for monocytes, neutrophils and blood leukocytes and produces chemoattractants that attract monocytes to the vascular intima. Monocytes migrating to the subendothelial space differentiate into macrophages, which act as catalysts for the formation of oxidized low density lipoproteins (LDL), migration and proliferation of smooth muscle cells from the muscle membrane into the intima. Particles of modified LDL are preferably captured by specific receptors by macrophages, which are transformed into foam cells. Activated macrophages that produce metalloproteinases and collagenase, in certain situations, contribute to the breakdown of collagen in the plaque and the emergence of such a formidable complication as plaque rupture, thrombus formation and the development of myocardial infarction. Thus, it has been substantiated that macrophages play a key role in the development of atherosclerotic lesions (see MI Dushkin. Macrophages and atherosclerosis: pathophysiological and therapeutic aspects. // Bul. SB RAMS, (2006); No. 2 (120), p. .47-55). It has been proved that phospholipases are biomarkers of the activity of the atherosclerotic process, the effectiveness of therapy both in patients with atherosclerosis and in patients with complications of atherosclerosis, in particular ischemic heart disease - coronary heart disease (angina pectoris II FC) (see SN Bogdanova. Clinical experimental study of the influence of the nutritional factor on the activity of lysosomal hydrolases of platelets and mononuclear leukocytes in atherosclerosis. Abstract of the thesis. Cand., (1992), 28 pp .; EN Dankovtsev, DA Zateishchikov Biomarkers in cardiology: lipoprotein-associated phospholipase A2 // Pharmateca (2007); №15 (149) Cardiology, Neurology, pp. 22-28).

The lipid profile is largely determined by the state of macrophages, their enzymatic reactions. In this regard, research and development of agents that change the activity of macrophage lipases, in particular, acid phospholipase A1 (cFLA1) (EC 3.1.1.32) at an optimal pH of 4.0, causing degradation of the phospholipid structure, cleaving off acyl residues from carbon atoms C ₁ per molecule of lecithin and other phospholipids, to form an intermediate hydrolysis products - and lysophospholipids lysolecithin having strong membranotoksicheskim action (see AA Basil, VA Tutelian Lysosomes -... Science, 1976, 380 .; X. Brokerhof , R. Jensen Lipolytic enzymes, translated from English, M., 1978, p. 242-356). In the literature there is information about the possibility of a decrease in the total (total) activity of cFLA1 by various chemical compounds (see NB Gubergritz. "Essentiale forte N""EssentialeN" in hepatology and gastroenterology. Suchasna gastroenterology, No. 5 (43), 2008 r . pp. 79-89). However, there is a need to search for and put into practice new diagnostic and pharmaceutical agents with the indicated activity.

The revealed change in the activity of cFLA1 can be considered as a compensatory reaction of enzyme systems against the background of the prevalence of processes of nonspecific unregulated endocytosis of modified low density lipoproteins (LDL) or supramolecular LDL-containing complexes, when the specific receptor-mediated pathway of endocytosis of LDL is inhibited by the principle of eutheriol cholesterol (see Pogozheva A.V. Cardiovascular diseases, M., 2005, 205 p.). In this case, under conditions of substrate saturation, a relative deficiency of certain lysosomal enzymes that break down ECS may occur, which leads to the accumulation of ECS and triglycerides in cells and an increased risk of atherosclerosis (see Nagornev V.A. Pathogenesis of atherosclerosis. St. Petersburg: "Chromis", 2006) ...

Thus, the influence of the reaction of acidic phospholipase A1 on the activity of subcellular structures during the development of atherosclerosis necessitates the search for and introduction into practice of new diagnostic and medicinal agents capable of changing the activity of microphage lipases.

As closestanalogue a method for reducing the total activity of cFLA1 using a complex of tris- (2-hydroxyethyl) amine with bis- (2-methylphenoxyacetate) zinc (see Rasulov M.M., Voronkov M.G., Storozhenko P.A., Mirskova A.N., Snisarenko T.A., Abzaeva K.A., Vaganov M.A. The use of a complex of tris- (2-hydroxyethyl) amine with bis- (2-methyl-phenoxyacetate) zinc to reduce the total activity of acidic phospholipase A1 // Invention patent RU No. 2545888, 10.04.2015).

Complex of tris- (2-hydroxyethyl) amine with bis- (2-methylphenoxyacetate) zinc (analog), or citrimine (zincatran) ,

has the formula: (HOCH ₂ CH ₂ ) ₃ N⋅Zn (OOCCH ₂ OC ₆ H ₄ CH ₃ ^-2 ) ₂

and view:

The disadvantage of the prototype can be attributed to the small effect of inhibition of phospholipase activity.

The claimed invention relates to the priority direction of development of science and technology "Biomedical and veterinary technologies for life support and protection of humans and animals" [see. Alphanumeric Index to the International Patent Classification in Priority Areas of Science and Technology Development / Yu.G. Smirnov, E.V. Skidanova, S.A. Krasnov - M .: PATENT, 2008. - p. 15].

The objective of the present invention is to develop a new agent that can be used to reduce the overall activity of the lysosomal lipolytic enzyme, acid phospholipase A1.

The technical result is an expansion of the arsenal of methods of influencing the activity of acidic phospholipase A1 by using the agent - 1- (germatran-1-yl) -1-hydroxyethylamine, due to its newly identified biological activity, namely, the ability to reduce the total activity of cFLA1.

The problem is solved by the fact that as a means that lowers the total activity of acid phospholipase A1, it is proposed to use the previously synthesized (see Voronkov M.G., Adamovich S.N. et al. Synthesis of new biologically active O-hydrometallatranes // ZhOKh, 2009 , v.79, No. 1, S. 162-163), a biologically active compound - 1- (germatran-1-yl) -1-hydroxyethylamine, which has the formula:

,

,

1- (germatran-1-yl) -1-hydroxyethylamine has the following form:

It is known that 1- (germatran-1-yl) -1-hydroxyethylamine has an adaptogenic effect [see. Zhigacheva I.V. Binyukov V.I., Mil E.M., Generozova I.P., Rasulov M.M. The effect of organogermanium compounds on the functional state of mitochondria of plant and animal origin // Scientific Almanac (Biological Sciences) 2015, No. 7 (9), 955-966].

1- (germatran-1-yl) -1-oxyethylamine can be used in clinical medicine to create drugs with antihyperlipidemic action and no side effects.

The claimed biological activity of 1- (germatran-1-yl) -1-hydroxyethylamine as an agent with the property of reducing the activity of acidic phospholipase A1 was not known and was not described in the literature.

It was shown for the first time that the introduction of 1- (germatran-1-yl) -1-hydroxyethylamine reduces the activity of the lysosomal lipolytic enzyme cFLA1.

Thus, there is proposed a method for reducing the total (total) activity of a lysosomal lipolytic enzyme - acid phospholipase A1, including the introduction of a composition that contains 1- (germatran-1-yl) -1-hydroxyethylamine as an active substance, as well as a pharmaceutically acceptable aqueous carrier, to an experimental animal intramuscularly daily for 2 months at a dose of 5.0 mg / kg of the active substance.

In this case, the use of official distilled water, water for injection or an official physiological solution - 0.9% sodium chloride solution is proposed as a pharmaceutically acceptable aqueous carrier.

In this case, the solution is prepared "ex tempore" and may contain 5-20 wt.% 1- (germatran-1-yl) -1-hydroxyethylamine.

During the development of the invention, the authors used various solvents to obtain an agent for influencing the activity of cFLA1. Various water-based solutions were tested, in particular a solution of the above active substance in water for injection, official distilled water and in official 0.9% sodium chloride solution. Moreover, in all cases, similar results were obtained. It should be noted that we studied the solutions obtained in this way with different concentrations of the active substance - 1- (germatran-1-yl) -1-hydroxyethylamine (5%, 10%, 20%, 50%, 70%). At the same time, the positive effects from their use, noted below, did not differ significantly. Studies have used different doses active substance: from 0.1 mg / kg to 20 mg / kg of animal weight. The effect of inhibiting the activity of the cFLA1 enzyme was present in all cases and was directly proportional to the dose of the active substance used. Accordingly, in order to achieve the technical result indicated by us, it is important as such a new property of this compound, and not its dose or a specific aqueous solvent. Therefore, in the example below, only the experiment using the active substance at a dose of 5.0 mg / kg daily is given for demonstration. In this case, a solution of the active substance was used, which was prepared ex tempore using an official saline solution (0.9% sodium chloride), mixing the active substance with it.

The possibility of carrying out the invention can be illustrated by the following data.

Example

The experiments were carried out on Chinchilla rabbits with an initial body weight of 1.8-2.0 kg in accordance with the “Rules of laboratory practice in the Russian Federation”. The rules are approved by order of the Ministry of Health of the Russian Federation dated June 19, 2003 No. 267 (Rules of laboratory practice in the Russian Federation of the Ministry of Health of the Russian Federation, Order No. 267 dated June 19, 2003 http://www.kodeks.ru (April 24, 2010)). The animals were kept in accordance with the rules of the European Convention for the Protection of Vertebrate Animals used for experimental and other scientific purposes. At the end of the experiment, the animals were euthanized by cervical dislocation of the 5th vertebra, observing the rules of the "European Convention for the Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes" (Strasbourg, 1986).

1- (germatran-1-yl) -1-hydroxyethylamine was mixed "ex tempore" until complete dissolution in an official 0.9% sodium chloride solution.

The complex of tris- (2-hydroxyethyl) amine with bis- (2-methylphenoxyacetate) zinc (analogue - citrimine) was mixed "ex tempore" until complete dissolution in the official 0.9% sodium chloride solution.

Hyperlipidemia was induced by the Anichkov-Khalatov method (a well-known experimental model for the development of atherosclerosis).

The animals were grouped by 10 heads as follows:

1) the group of the experiment, which was injected intramuscularly with a freshly prepared solution of 1- (germatran-1-yl) -1-oxyethylamine at a dose of 5.0 mg / kg of the active substance of the animal weight daily for 2 months.

2) a placebo-control group, which was injected intramuscularly with an equal volume of the official 0.9% sodium chloride solution daily for 2 months.

3) a group of animals that received intramuscularly a freshly prepared solution of an analogue - cytrimine at a dose of 5.0 mg / kg of the active substance of the animal weight daily for 2 months.

The reference was the data obtained from intact rabbits.

The animals were deprived of food 12 hours before slaughter.

The abdominal cavity was opened under thiopental anesthesia, thoroughly perfusing the liver with chilled saline (0.9% sodium chloride) before isolation. Homogenization of the liver was carried out in a glass Potter homogenizer for 90 s at a pestle rotation speed of 200 g with a translational movement of the glass beaker of the homogenizer up and down. A 0.25M sucrose solution pH 7.4 with 1 mM EDTA was used as a suspending medium. The final dilution of the liver homogenates was 1:20 (w / v). The supernatant was used to further investigate the activity of cFLA1. The PLA1 activity was determined by a radiometric method (see W. Stoffel, U. Trabert, Studies on the occurrence and properties of lisosomal phospholipases A1 and A2 and the degradation of phosphatidic acid in rat liver lysosomes // Hoppe Seylers Z. Fhysiol. Chem. - 1969 - Bd. 350. - S. 836-844), using the previously synthesized 1-acyl-2 (1-14C) -oleoyl-glycero-3, sn-phosphorylcholine (AF Robertson, WEM Leuds Positional specificities in phospholipid hydroly-ses // Biochemistry (Wash.) - 1962. - Vol. 1 - P. 804-810). The incubation mixture contained 150 nM labeled lecithin, 160 μM 0.1 N acetate buffer (pH 4.5) and a source of enzymes in a final volume of 1.3 ml. The incubation was carried out on a water vibrator for 30 min at 37 ° C. The reaction was stopped by adding 3 ml of a mixture of chloroform: methanol (1: 2 v / v) and immediately extracted by the method of Klayer and Dyer. The reaction products were separated by thin-layer chromatography in a fixed layer of silica gel on glass plates 20/20 cm in size in a solvent system chloroform: methanol: water (65: 25: 4). Fractions of lysolycetin, lecithin, and fatty acids were extracted with a mixture of chloroform: methanol at a volume ratio of 1: 2. The extracts were placed in vials with 10 ml of scintillation fluid containing 5 g of 2,5-diphenyloxazole and 300 mg of 1,4 bis / 2- (5 phenyloxazolyl) -benzene in 100 ml of high-purity toluene. Radioactivity was measured on a scintillation counter "Rackbeta 1215" LKB (Sweden). The activity of PLA1 was judged by the formation of labeled lysolycetin resulting from the action of these enzymes on 1-acyl-2 (1-14C) -oleoyl-glycero-3, sn-phosphorylcholine. The activity of cFLA1 was expressed in μM of the product per min / 1 g of protein.

Statistical processing of the data was carried out by the Student's method. Data were presented as mean and standard error values - M and m , respectively. For relative values, 95% confidence intervals were calculated. Differences were considered significant at p ≤ 0.05 (see Petri A., Sebin K., 2009).

Results .

It was revealed that the use of 1- (germatran-1-yl) -1-oxyethylamine (in the form of a solution) in the liver tissue decreases the level of cholesterol and total lipids (table 1).

Comparative analysis of the data in Table 1 shows that the increase in the activity of cFLA1 in the liver of the control group of rabbits exposed to artificial hyperlipidemia significantly exceeds the reference values.

Correlation analysis revealed a relationship between the level of cFLA1 activity and lipid metabolism indicators after administration of 1- (germatran-1-yl) -1-hydroxyethylamine solution at a dose of 5 mg / kg of the active substance of the animal weight for 2 months (Table 2).

The use of cytrimin (analogue) also leads to a decrease in FLA1. However, daily use for 2 months of 1- (germatran-1-yl) -1-hydroxyethylamine at a dose of 5 mg / kg of the animal weight of the active substance causes a more pronounced decrease in lipid metabolism, therefore, confirms its higher activity compared to citrimine ( analogue) to reduce the overall activity of acidic phospholipase A1 - an enzyme that demonstrates the activity of the atherosclerotic process.

The invention will make it possible to create, on the basis of 1- (germatran-1-yl) -1-oxyethylamine, new pharmacological preparations for lowering the total activity of acidic phospholipase A1 and preventing atherosclerotic lesions of blood vessels.

Table 1.

Lipid metabolism indicators when using 1- (germatran-1-yl) -1- oxyethylamine

Indicators Liver Standard - intact Total lipids (mM / l) 0.22 ± 0.013 Cholesterol (mM / L) 0.12 ± 0.01 Triacylglycerols (mM / L) 0.36 ± 0.01 β-lipoproteins (mM / l) 0.2 ± 0.002 cFLA1 (μM / min / per 1 g of protein) 0.9 ± 0.04 Placebo control Total lipids (mM / l) 1.4 ± 0.09 ** Cholesterol (mM / L) 1.5 ± 0.9 ** Triacylglycerols (mM / L) 1.1 ± 0.1 ** β-lipoproteins (mM / l) 1.8 ± 0.1 ** cFLA1 (μM / min / per 1 g of protein) 4.1 ± 0.2 ** Analogue - citrimine at a dose of 5 mg / kg Total lipids (mmol / l) 0.6 ± 0.05 * Cholesterol (mmol / L) 0.26 ± 0.05 * Triacylglycerols (mmol / L) 0.5 ± 0.067 * β-lipoproteins (mmol / l) 0.36 ± 0.002 * cFLA1 (μM / min / per 1 g of protein) 1.3 ± 0.1 * Experience - 1- (germatran-1-yl) -1-hydroxyethylamine at a dose of 5 mg / kg Total lipids (mM / l) 0.3 ± 0.012 * Cholesterol (mM / L) 0.21 ± 0.03 * Triacylglycerols (mM / L) 0.26 ± 0.05 * β-lipoproteins (mM / l) 0.3 ± 0.02 * cFLA1 0.95 ± 0.03 *

Note : * -p <0.05 in relation to control;

** - p <0.05 in relation to the standard.

Table 2 .

Correlation coefficients ( r ) of the activity of acidic PLA1 in the liver with indicators of lipid metabolism when using 1- (germatran-1-yl) -1-oxyethylamine at a dose of 5 mg / kg

Enzyme Common lipids Cholesterol Triacil -
glycerin β - lipo-proteins cFLA1 r = 0.39 r = 0.37 r = 0.44 r = 0.47

Claims (4)

1. A method for reducing the total (total) activity of a lysosomal lipolytic enzyme, acid phospholipase A1, comprising administering a drug to an animal, characterized in that a composition containing 1- (germatran-1-yl) -1-hydroxyethylamine is used as such a drug and pharmaceutically an acceptable aqueous carrier, which is administered intramuscularly to the animal at a dose of 5.0 mg of active substance / kg of animal weight daily for 2 months. 2. A method according to claim 1, characterized in that a composition obtained ex tempore by mixing 1- (germatran-1-yl) -1-hydroxyethylamine and a pharmaceutically acceptable aqueous carrier is used. 3. A method according to any one of claims. 1 or 2, characterized in that water for injection, official distilled water or official physiological saline is used as the aqueous carrier. 4. The method according to claim 3, characterized in that the composition may contain 5-20 wt% 1- (germatran-1-yl) -1-hydroxyethylamine.