RU2769051C1 - Peptide that improves the functional properties of the vascular endothelium, a pharmaceutical composition based on it and the method for its application - Google Patents

Abstract

FIELD: pharmacology.

SUBSTANCE: group of inventions relates to the field of biochemistry and pharmacology, namely to drugs based on peptides that improve the functional properties of the vascular endothelium in their violation of various genesis, including atherosclerosis. The use of the peptide formula β-aspartyl-alanine H-Asp(Ala)-OH is proposed to improve the functional activity of the vascular endothelium, as well as in the prevention and/or treatment of endothelial dysfunction, for example, such as endothelial dysfunction in atherosclerosis, restenosis, coronary heart disease, cerebral circulation disorders.

EFFECT: expansion of the range of medicines based on peptides to improve the functional properties of the vascular endothelium.

3 cl, 2 dwg, 1 tbl, 6 ex

Description

SUBSTANCE: group of inventions relates to the field of biochemistry and pharmacology, namely to drugs based on peptides that improve the functional properties of the vascular endothelium in their disorders of various origins, including atherosclerosis.

Cardiovascular diseases associated with atherosclerosis and restenosis are the main causes of disability and death in developed countries. According to the World Health Organization, they threaten to become the leading cause of death in developing countries in the next decade. Atherosclerosis is a chronic inflammatory metabolic disease that develops with a violation of complex interactions between several types of cells in the vascular wall [Gavrilova N.E., Metelskaya V.A., Perova N.V., Yarovaya E.B., Mazaev V.P. , Urazalina S.Zh., Boytsov S.A. The relationship between the severity of coronary atherosclerosis, risk factors and markers of atherosclerotic lesions of carotid and peripheral arteries. Cardiovascular therapy and prevention. 2013. V. 12. No. 1. S. 40-45]. One possible cause of atherosclerosis is endothelial dysfunction. Currently, drugs from the group of statins, such as Lovastatin, Atorvastatin and others, are used to treat vascular atherosclerosis [VIDAL Drug Reference (Reg. No: P N012626/01 dated 23.08.10) http://www.vidal.ru/] . All of these statin-based drugs have a similar mechanism of action and side effects. Lovanstatin is a lipid-lowering agent from the group of statins, an inhibitor of HMG-CoA reductase. It is a prodrug, since it has a closed lactone ring in its structure, which is hydrolyzed after ingestion. The lactone ring of statins is similar in structure to part of the enzyme HMG-CoA reductase. According to the principle of competitive antagonism, the statin molecule binds to the part of the coenzyme A receptor where this enzyme is attached. Another part of the statin molecule inhibits the conversion of hydroxymethylglutarate to mevalonate, an intermediate in the synthesis of the cholesterol molecule. Inhibition of the activity of HMG-CoA reductase leads to a series of successive reactions, as a result of which the intracellular cholesterol content decreases and a compensatory increase in low-density lipoprotein (LDL) activity occurs and, accordingly, cholesterol catabolism (Xc) is accelerated. The lipid-lowering effect of statins is associated with a decrease in the level of total cholesterol. However, at present, the leading hypothesis for the development of atherosclerosis is endothelial dysfunction, while impaired metabolism of lipoproteins, including LDL, is interpreted as a secondary manifestation of this disease. Thus, drugs from the statin group cannot eliminate endothelial dysfunction, which is the cause of atherosclerosis. In addition, the use of Lovastatin has a number of side effects on the cardiovascular, central nervous, digestive, musculoskeletal, reproductive and excretory systems. In this regard, the search for new effective and safe treatments for cardiovascular diseases is relevant.

The present invention is aimed at solving the problem of obtaining a peptide having the ability to improve the functional activity of the vascular endothelium, manifested in the activation of proliferation, the protective properties of endothelial cells, the reduction of apoptosis, and the use of this peptide for the manufacture of a pharmaceutical composition that improves the functional properties of the vascular endothelium.

The technical result of the invention lies in the fact that it is proposed to use the β-aspartyl-alanine peptide of the formula H-Asp(Ala)-OH to improve the functional activity of the vascular endothelium, as well as to manufacture a pharmaceutical composition that improves the functional activity of the vascular endothelium by regulating proliferation, apoptosis and functional properties of vascular endothelial cells.

It was found that the peptide β-aspartyl-alanine of the formula H-Asp(Ala)-OH has vasoprotective biological activity due to the activation of proliferation, the protective properties of endothelial cells, and the reduction of apoptosis, while the action of the peptide has a geroprotective character, expressed in the prevention of atherosclerotic changes in the endothelium . It was also found that the use of the H-Asp(Ala)-OH peptide neutralizes the effect of cytotoxic substances on vascular endothelial cells.

It is proposed to use the β-aspartyl-alanine peptide of the formula H-Asp(Ala)-OH for the preparation of a drug that improves the functional properties of the vascular endothelium.

Another aspect of the present invention relates to a pharmaceutical composition that improves the functional properties of the vascular endothelium, containing as an active principle an effective amount of a β-aspartyl-alanine peptide of the formula H-Asp(Ala)-OH and a pharmaceutically acceptable carrier.

When this pharmaceutical composition is in a form suitable for parenteral, oral, intranasal or topical administration.

The next aspect of the present invention relates to a method for the prevention and treatment of endothelial dysfunction, including vascular atherosclerosis, which consists in administering to a patient a pharmaceutical composition containing a peptide of the formula H-Asp(Ala)-OH as an active principle, in doses of 0.1-10 μg /kg body weight at least once a day when administered intramuscularly, 1-10 mcg/kg body weight at least once a day when administered intranasally and 10-100 mcg/kg body weight at least twice a day when oral administration during the period necessary to achieve a therapeutic effect - at least 30 days.

Peptide β-aspartyl-alanine of the formula H-Asp(Ala)-OH is obtained by the classical method of peptide synthesis in solution.

The regulatory effect of the H-Asp(Ala)-OH peptide on proliferation, apoptosis, and functional activity of vascular endothelial cells was revealed during its experimental study.

The study of biological activity was carried out in organotypic and dissociated cultures of vascular endothelium of rats and humans in normal conditions, with aging and atherosclerosis.

The concept of "pharmaceutical composition" means various dosage forms containing an effective amount of the H-Asp(Ala)-OH peptide, which can be used in medicine as a means of stimulating the function of the vascular endothelium.

To obtain pharmaceutical compositions of the present invention, an effective amount of the H-Asp(Ala)-OH peptide as an active principle is mixed with pharmaceutically acceptable carriers according to compounding methods adopted in pharmaceuticals [see, for example, Gavrilov A.S. Pharmaceutical technology. Manufacturing of medicines / A.S. Gavrilov - M.: GEOTAR-Media, 2016. - 760 p.; Krasnyuk I.I. Pharmaceutical technology. Technology of dosage forms: textbook / I.I. Krasnyuk, G.V. Mikhailova, L.I. Muradov. - M.: GEOTAR-Media, 2016. - 560 p.; Manufacturing technology of dosage forms: textbook / V. A. Grossman - M.: GEOTAR-Media, 2018. - 336 p.; Pharmaceutical technology. Manufacturing of medicines [Electronic resource]: textbook. allowance / Loyd V. Allen, A.S. Gavrilov. - M.: GEOTAR-Media, 2014. - 512 p.; Quality control of medicines [Electronic resource]: textbook / ed. T.V. Pleteneva - M.: GEOTAR-Media, 2014. - 560 p.; Pharmaceutical technology. Technology of dosage forms [Electronic resource]: textbook / I.I. Krasnyuk, G.V. Mikhailova, L.I. Muradov. - M.: GEOTAR-Media, 2013. - 560 p.].

The concept of "effective amount" means the use of such an amount of the active principle, which, in accordance with its quantitative indicators of activity and toxicity, and also on the basis of the knowledge of a specialist, should be effective in dosage form.

The carrier may take various forms, which depend on the dosage form of the drug desired for administration to the body.

The essence of the invention is illustrated by the following graphics:

Figure 1 shows the effect of the H-Asp(Ala)-OH peptide on the expression of the Ki67 proliferation marker in human umbilical cord cell culture, passage 3: A - control, B - H-Asp(Ala) -OH peptide, immunocytochemical staining with hematoxylin staining of nuclei , x 200.

Figure 2 shows the effect of the H-Asp(Ala)-OH peptide on the expression of the Cx43 marker in a culture of human umbilical cord endothelial cells, passage 3: A - control, B - H-Asp(Ala) -OH peptide, immunocytochemical staining with hematoxylin staining of nuclei , x 400.

The invention is illustrated by an example of the synthesis of the β-aspartyl-alanine peptide of the general formula H-Asp(Ala)-OH (example 1), an example of a toxicity test (example 2), an assessment of the biological activity and mechanism of action of the peptide in cultures of rat and human endotheliocytes in normal conditions, at aging and atherosclerosis (examples 3-4), demonstrating its pharmacological properties and confirming the possibility of obtaining a preventive and / or therapeutic effect (example 5), an example of the effectiveness of the use of a pharmaceutical composition based on the β-aspartyl-alanine peptide of the formula H-Asp (Ala) - OH to improve vascular function in patients with cerebrovascular disease (example 6).

Example 1 . Synthesis of the peptide H-Asp(Ala)-OH

Compound name: β-aspartyl-alanine

Structural formula: H-Asp(Ala)-OH

Molecular formula: C ₇ H ₁₂ N ₂ O ₅

Molecular weight without counterion: 204.18

Counterion - acetate

Appearance: Odorless white amorphous powder

Method of synthesis: the peptide was obtained by the classical method of synthesis in solution according to the scheme:

Characteristics of the finished product

Solubility: soluble in water, isotonic sodium chloride solution 0.9%; insoluble in alcohol 95%, chloroform, ether and other organic solvents.

Transparency and color of the solution: a solution of 0.05 g of the drug in 10 ml of water is transparent and colorless.

- pH 0.001% solution: 4.0-5.0 (potentiometrically);

- moisture content: no more than 10%;

- specific optical rotation: [α] _D ²² : from -50 ° to -54 ° (c=1, H ₂ O);

- the content of the main substance according to HPLC: not less than 97%;

- thin layer chromatography: TLC-individual, R _{f =} 0.82 (acetonitrile-water 1:3).

Synthesis Example

1. Z-Asp(OSu)-OH (I), β-N-oxysuccinimide ester of N-benzyloxycarbonyl(α-benzyl)-aspartic acid. 2.21 g (6.2 mmol) of finely divided powder of N-benzyloxycarbonyl(α-benzyl)-aspartic acid are dissolved in 50 ml of dimethylformamide and 0.71 g (6.2 mmol) of finely divided powder of N-oxysuccinimide are added. 1.27 g (6.2 mmol) of N,N'-dicyclohexylcarbodiimide powder are dissolved in 20 ml of dimethylformamide. Both solutions are cooled to -15°C. The solutions are combined and stirred under ice-cooling for 24 hours. The resulting activated ester solution is used in the next step.

2. Z-Asp(Ala-OBzl)-OBzl (II), N-benzyloxycarbonyl-(β-ala-nilbenzilate)-aspartic acid α-benzyl ester. To a solution of the activated ester Z-Asp(OSu)-OBzl (I) obtained in the previous step, 1.34 g (6.2 mmol) of finely divided powder of alanine benzyl ester tosylate and 0.86 ml of triethylamine are added. Stir the reaction mixture at room temperature for 2 days. Then, 150 ml of a 2 N sulfuric acid solution are gradually poured into the reaction mixture with stirring, the product precipitates in the form of an oil, which is then dissolved in 50 ml of ethyl acetate.

Then the contents of the reaction flask are stratified in a separating funnel, the lower aqueous phase is poured into a beaker with a capacity of 250 ml, and the upper organic phase is poured into a beaker with a capacity of 500 ml. The procedure is repeated three times with new portions of ethyl acetate (30 ml each), then all ethyl acetate portions are combined in the original separating funnel, into which 20 ml of 2 N sulfuric acid solution is added and extraction is carried out, repeating the procedure twice. The ethyl acetate solution is then washed successively with water (30 ml), twice with sodium bicarbonate solution (30 ml), twice with sulfuric acid solution (30 ml) and water (30 ml). Then about 20 mg of anhydrous sodium sulfate is added to the ethyl acetate solution and left for 10-12 hours.

The filtered ethyl acetate solution is evaporated in vacuum at a temperature not exceeding 40 ° C on a vacuum rotary evaporator (Buchi Rotavapor R-114 or similar) until the distillation of the solvent stops and a thick syrup forms at the bottom of the flask, which is dissolved in 20 ml of ethyl acetate, adding to the solution at constant stirring in portions of hexane until a stable turbidity appears (about 20 ml). The flask with the obtained cloudy system is left in the refrigerator at 4°C for at least 2 days to complete the crystallization process.

The precipitated crystalline product is filtered under vacuum through a porous Schott filter, washed 2 times with hexane (5 ml), then the product is transferred to a Petri dish without a lid. Finally, the product is dried over phosphorus pentoxide in vacuum at a temperature not exceeding 40°C to constant weight.

3. H-Asp(Ala)-OH (III), β-aspartyl-alanine. The α-benzyl ester of N-benzyloxycarbonyl-(β-alanylbenzilate)-aspartic acid (II) obtained in the previous step is dissolved in a mixture of methanol (60 ml), distilled water (20 ml) and acetic acid (20 ml) in the presence of 3 ml of palladium-on-carbon (Pd/C) catalyst.

Control over the completeness of deblocking is carried out in the TLC mode on Silufol plates in the benzene-acetone system (2:1), the deblocked product has zero mobility, the initial product has Rf=0.86. The release reaction is considered complete if there are only trace amounts of the original product (visually).

The combined filtrate is evaporated in vacuo at a temperature not exceeding 40°C.

For purification, 290 mg of the drug is dissolved in 4 ml of 0.01% trifluoroacetic acid and subjected to high performance liquid chromatography on a reverse phase column 50 × 250 mm Diasorb-130-C16T, 7 μm. Beckman System Gold Chromatograph, 126 Solvent Module, 168 Diode Array Detector Module. Chromatography conditions - A: 0.1% trifluoroacetic acid; B: 50% acetonitrile in 0.1% trifluoroacetic acid; gradient B 0 → 5% in 80 min. Sample volume 5 ml, detection at 215 nm, scanning at 190-600 nm, flow rate 10 ml/min.

Select fractions of the main peak, free from impurities (in the approximate range of 48.0 min - 60.0 min), combine them and evaporate in vacuum until a syrup is formed, repeating the evaporation 5 more times each time with a new portion (10 ml) of 10% acetic acid. The residue in the flask is dried in vacuum at a temperature not exceeding 40°C over solid KOH for at least 2 days. After drying, the product should be odorless.

After evaporation and drying in vacuo, the residue is taken up in 20 ml of deionized water and lyophilized.

4. Analysis of the finished product

- The content of the main substance is determined by HPLC on a Phenomenex C 18 LUNA 4.6×150 mm column. A: 0.1% TFA, B: MeCN; gradient B 0-100% in 10 min. Flow rate 1 ml/min. Detection at a wavelength of 220 nm, scanning at wavelengths of 190-600 nm, sample 20 µl. The content of the main substance is not less than 97%.

- TLC: on the chromatograms of samples treated with a solution of ninhydrin, only two spots (preparation and GSO) of purple color with matching R _f are allowed; on the chromatograms of samples treated with a solution of benzidine, the presence of only two spots (preparation and GSO) of dark blue color with matching R _f is allowed (acetonitrile-water 1: 3, PTSH-P-V-UV Sorbfil plates, silica gel STX-1VE 8- 12 µm, chlorine/benzidine development).

- Moisture content: no more than 10% (gravimetrically by loss of mass on drying 20 mg at 100 °C).

- pH 0.01% solution: 4.0 to 5.0 (potentiometrically).

- Specific optical rotation: from -50° to -54° in terms of dry matter (0.1% aqueous solution, temperature 23 °C, D-line of the sodium spectrum).

Example 2 . Study of the toxicity of the peptide H-Asp(Ala)-OH

The general toxic effect of the peptide was studied in accordance with the requirements of the “Guidelines for conducting preclinical studies of drugs. FSBI "NCESMP" of the Ministry of Health and Social Development of Russia. Part 1” (Moscow, Grif and K, 2012. 944 p.) acute toxicity with a single administration of the drug, as well as chronic toxicity with prolonged administration of the peptide.

The study of acute toxicity was carried out on 110 outbred male mice weighing 20-24 g. The animals were randomly divided into 11 equal groups of 10 animals in each group. The drug was administered to animals once intramuscularly or intragastrically (through a tube) at doses of 1 mg/kg, 2 mg/kg, 3 mg/kg, 4 mg/kg, 5 mg/kg (several thousand times higher than the recommended therapeutic dose (TD)). for clinical study, which is 0.0014 mg / kg) in a volume of 0.25 ml of isotonic 0.9% sodium chloride solution. Animals of the control group were injected with 0.9% NaCl solution in the same volume.

Within 72 hours and then after 14 days, no death of mice was detected in any group of animals. There were no changes in the general condition, behavior, physical activity, hair and skin, physiological functions of animals.

Thus, the dipeptide H-Asp(Ala)-OH at doses several thousand times higher than the therapeutic recommended for clinical trials does not cause acute toxic reactions when administered intramuscularly or intragastrically, which indicates a greater therapeutic breadth of the drug.

The chronic toxicity of the dipeptide H-Asp(Ala)-OH, obtained by the claimed method, was studied during its long-term administration to rats weighing 150-250 mg. Animals were injected daily intramuscularly or intragastrically (through a tube) with the drug at doses of 0.1 mg/kg (70 TD), 1.0 mg/kg (700 TD), 3 mg/kg (2000 TD) in 0.5 ml of saline within 30 days. Animals were observed for 60 days after the start of the experiment. Animal behavior, food and water consumption, the condition of the hairline and mucous membranes were noted. Animals were weighed weekly. At the beginning and at the end of the experiment, hematological and biochemical studies were performed. The functions of the cardiovascular system, liver, pancreas, kidneys and adrenal glands were evaluated. After the end of the administration of the drug, the animals were subjected to pathomorphological examination in order to assess the state of various parts of the brain and spinal cord, heart, aorta, lungs, liver, kidneys, organs of the endocrine and immune systems.

When assessing the general condition of animals, morphological and biochemical parameters of peripheral blood, the morphological state of internal organs, the state of the cardiovascular and respiratory systems, liver and kidney function, no pathological changes were found in the body.

The study of subacute and chronic toxicity of the dipeptide H-Asp(Ala)-OH indicates the absence of side effects with prolonged use of the drug intramuscularly or intragastrically at doses exceeding the therapeutic dose by 70-2000 times.

Example 3 . Effect of H-Asp(Ala)-OH Peptide on Proliferation, Apoptosis, and Activation of Protective Properties of Rat Vascular Endotheliocytes in Organotypic and Dissociated Cultures

Organotypic cultivation of vascular tissue (aorta) of young 3-month-old (n=60) and old 20-month-old Wistar rats (n=60) was carried out in a nutrient medium of the following composition per 100 ml: Hank's solution 38 ml, Eagle's medium 36 ml , fetal bovine serum 25 ml, glucose 40% - 1.0 ml, gentamicin 100 U / ml. After removing the vessels, they were placed in a sterile Petri dish and divided into explants (fragments about 1 mm ³ in size). The explants were cultivated for 3 days in 3 ml of nutrient medium in a CO2 incubator at a temperature _of 36.7°C in a medium with ₅ % CO2 content. Peptide H-Asp(Ala)-OH was added to the cultures at a concentration of 0.05 ng/ml, because previously it proved to be the most effective in this experiment. A similar amount of nutrient medium was added to control cultures. To simulate the cytotoxic effect, cyclophosphamide (CP) was used, which was added to cultures at a concentration of 2 mg/mL.

In explants of vessels after 3 days of cultivation, two zones were determined: central and peripheral. The central zone was represented by cells initially placed on a collagen substrate. The peripheral zone (growth zone) consisted of newly formed cells that had settled around the circumference. To quantify the growth of explants, the area index (AI) was determined. This is the ratio of the area of the entire explant to the area of the central zone.

A dissociated vascular cell culture was obtained from the aorta of a young Wistar rat (3 months old). Aortic pieces were exposed to 0.2% collagenase II for 10 minutes at 37°C in an incubator and centrifuged for 5 minutes at 400 g. Cells were supplemented with 2 ml of RPMI medium supplemented with 20% fetal bovine serum, HEPES 1.5%, and antibiotics 50,000 U/vial penicillin G and 50 mg/vial streptomycin. Cells were grown in 50 ml surface-treated flasks in 5 ml culture medium (20% fetal bovine serum, 82.5% RPMI, 1.5% HEPES, L-glutamine) at 36.7°C in medium with 5% CO content₂. After 5-7 days, when the primary culture reached the monolayer, it was subcultured at a concentration of 5x10³ cells per 50 ml vial. At each passage, the solution with peptides was added to the culture medium at a concentration of 100 ng/mL. Passaging was carried out after 3 days on the fourth, when the culture reached a monolayer. Cultivation was carried out up to 3 passages.

For immunocytochemical studies of organotypic and dissociated cultures of vascular cells, the cells were fixed with 96% alcohol. Blockade of endogenous peroxidase was carried out with 3% aqueous hydrogen peroxide solution for 15 min. Cell membranes were permeabilized with 0.5% Triton X100 solution. Then, primary monoclonal antibodies to CXCL12 (1:200, Vectorlab), Ki67 (1:150, Vectorlab), p53 (1:50, Dako) markers were used. Incubation with horseradish peroxidase conjugated secondary antibodies (EnVision Detection System, Peroxidase/DAB, Rabbit, Mouse) was performed for 30 minutes. To evaluate the results of immunocytochemical staining, a morphometric study was performed using a computer analysis system for microscopic images, consisting of a Nikon Eclipse E400 microscope, a Nikon DXM1200 digital camera, an Intel Pentium 4-based personal computer, and the Videotest-Morphology 5.2 program. In each case, 5 fields of view were analyzed at a magnification of 100. The expression area was calculated as the ratio of the area occupied by immunopositive cells to the total area of cells in the field of view and expressed as a percentage. This parameter characterizes the number of cells in which the studied marker is expressed.

Statistical data processing included the calculation of the arithmetic mean, standard deviation and confidence interval for each sample and was carried out in the Statistica program. To analyze the type of distribution, the Shapiro-Wilk test was used. To test the statistical homogeneity of several samples, nonparametric procedures of one-way analysis of variance (Kruskal–Wallis test) were used. The critical confidence level of the null hypothesis was taken equal to 0.05.

In organotypic cultures of vascular cells of young rats under the action of peptide H-Asp(Ala)-OH, PI significantly increased by 29±1% (p<0.05) compared with the control, and in organotypic cultures of vascular cells of old rats under the action of peptide H -Asp(Ala)-OH PI increased by 56±2% compared to the control. The introduction of CP into the culture medium led to a decrease in the IP of explants obtained from young and old rats by 16% and 18%, respectively. Simultaneous addition of CF and the H-Asp(Ala)-OH peptide to the organotypic cultures of vessels of young and old rats led to an increase in PI to the level of control values, which indicates a leveling of the cytotoxic effect of CF.

The H-Asp(Ala)-OH peptide did not affect the area of expression of the proliferation marker Ki67 in organotypic vessel cultures of young rats. In organotypic cultures of old rats, the H-Asp(Ala)-OH peptide doubled the expression area of Ki67. The results on the effect of the H-Asp(Ala)-OH peptide on the area index of the growth zone of vascular explants correlate with the data on the expression of the Ki67 protein, which is a marker of cell proliferation. Thus, the effect of the dipeptide H-Asp(Ala)-OH on the growth zone of vascular explants is associated with its stimulating effect on the expression of the Ki67 protein.

In organotypic vessel cultures of young rats, the H-Asp(Ala)-OH peptide increased the area of expression of the chemokine CXCL12 by 3.2 times compared to the control, and in organotypic vessel cultures of old rats, by 3.15 times compared to the control. The H-Asp(Ala)-OH peptide had no effect on the expression area of the proapoptotic p53 protein in organotypic vessel cultures of young and old rats.

In dissociated cultures of young rat vascular cells, the H-Asp(Ala)-OH peptide increased the expression area of the Ki67 proliferation marker by 96.7%. Peptide H-Asp(Ala)-OH increased the area of expression of the chemokine CXCL12 by 84.3% compared with the control in dissociated cultures of young rat vascular cells. The H-Asp(Ala)-OH peptide reduced the area of expression of the proapoptotic p53 protein by 164.5% compared with the control in dissociated cultures of young rat vascular cells.

Thus, the H-Asp(Ala)-OH peptide activates proliferation (increase in IP, Ki67 expression), reduces apoptosis (decrease in p53 expression), and activates the protective functions of endothelial cells (increased expression of the chemokine CXCL12) of rat vessels of different ages. In addition, the H-Asp(Ala)-OH peptide neutralizes the cytotoxic effect of CP on the organotypic culture of vessels in young and old rats. These data indicate the gero- and vasoprotective properties of the H-Asp(Ala)-OH peptide in relation to the vascular endothelium.

Example 4 Effect of H-Asp(Ala)-OH Peptide on Proliferation, Apoptosis, and Intercellular Contacts of Human Vascular Endotheliocytes

Primary cell cultures of human umbilical cord vessels were the objects for morphofunctional research. A fragment of a vein 0.1 cm in diameter was isolated from the umbilical cord and placed in a nutrient medium. To obtain primary cultures of endotheliocytes from a human umbilical cord vein, a modification of the standard method was used [Crampton S.P., Davis J., Hughes C.C. Isolation of human umbilical vein endothelial cells (HUVEC) // J Vis Exp. 2007. N3. P. 183]. Cells were grown up to passage 3 ("young" culture) and up to passage 14 ("old" culture). All cultures were divided into 2 groups: 1 - control (without adding the peptide), 2 - adding the peptide H-Asp(Ala)-OH at a concentration of 20 ng/ml. At each passage, the solution with the peptide was added to the growth medium at a concentration of 20 ng/ml. Passaging was carried out after 3 days on the 4th, when the cultures reached a monolayer. Cultivation was carried out up to passage 3 ("young" cultures) and up to passage 14 ("old" cultures), on which the cells were scattered on plates and immunocytochemical staining was performed. Primary cultures of endotheliocytes of human umbilical cord vessels by the 14th passage lost their ability to proliferate, and the level of apoptosis in them increased, in connection with which this particular passage was chosen as a model of "old" cells [Khokhlov A.N., Klebanov A.A., Karmushakov A.F., Shilovsky G.A., Nasonov M.M., Morgunova G.V. Testing of geroprotectors in experiments on cell cultures: the choice of an optimal model system. Bulletin of the Moscow University. Series 16: Biology. 2014. No. 1. S. 13-18]. Primary antibodies were used for immunocytochemical studies: Ki67 (mouse monoclonal antibodies to the human Ki67 protein, clone MIB-1, dilution 1:50, Dako, USA), p53 (mouse monoclonal antibodies to the human p53 protein, clone DO-7, dilution 1: 100, Dako, USA), Cx43 (rabbit polyclonal antibodies to human, rat and mouse Cx43 protein, clone GJA1, dilution 1:100, Abcam, England).

The Ki67 protein is a recognized and widely used proliferation marker. The aging process is characterized by the achievement of the Hayflick limit and a decrease or complete cessation of the ability of cells to divide. In this regard, the Ki67 protein can be an important marker for assessing the decrease in cell proliferative activity and the degree of involutive processes in the organ under study. The p53 protein is a transcription factor that acts as a suppressor of the formation of malignant tumors by activating apoptosis in body tissues. The p53 protein is activated upon DNA damage, as well as upon stimuli that can lead to such damage, or are a signal of cell aging and a violation of its functional activity. Increased expression of the p53 protein in the vascular endothelium is characteristic of cardiovascular pathology [Yokoyama M., Shimizu I., Nagasawa A., Yoshida Y., Katsuumi G., Wakasugi T., Hayashi Y., Ikegami R., Suda M., Ota Y., Okada S., Fruttiger M., Kobayashi Y., Tsuchida M., Kubota Y., Minamino T. p53 plays a crucial role in endothelial dysfunction associated with hyperglycemia and ischemia // J Mol Cell Cardiol. 2019 Vol. 129. P. 105-117]. Cx43, gap junction alpha-1 protein (GJA1), connexin 43. Connexin 43 belongs to the connexin family, which are components of gap junctions that form intercellular channels and ensure the diffusion of low molecular weight compounds between neighboring cells. Connexin 43 is expressed in vascular cells included in atherosclerotic plaques, including smooth muscle cells [Pfenniger A., Chanson M., Kwak B.R. Connexins in atherosclerosis. // Biochim Biophys Acta. 2013. Vol. 1828. N1. P. 157-166]. To evaluate the results of immunocytochemical staining, a morphometric study was performed using a computer analysis system for microscopic images and the Videotest Morphology 5.0 software. In each case, 10 fields of view were analyzed at a magnification of 200. The area of expression was calculated as the ratio of the area occupied by immunopositive cells to the total area of cells in the field of view and expressed as a percentage. Statistical processing of the experimental data included the calculation of the arithmetic mean, standard deviation, and confidence interval for each sample and was carried out using the Statistica 6.0 program. To analyze the type of distribution, the Shapiro-Wilk test was used. The critical confidence level of the null hypothesis was taken equal to 0.05.

In the control "old" cultures of endotheliocytes, the expression area of Ki67 was 1.6±0.1%, which is 2.6 times lower than in "young" cultures (4.2±0.4)%. Peptide H-Asp(Ala)-OH increased the area of expression of the proliferatropic protein Ki67 in "young" and "old" cultures of endotheliocytes by 1.3 and 1.9 times, respectively, compared with the control ("young" cultures are shown in Fig. 1) . In the control "old" cultures of endotheliocytes, the expression area of p53 was 4.5±0.5%, which is 3.8 times higher than in "young" cultures of endotheliocytes (1.2±0.1%). Peptide H-Asp(Ala)-OH reduced the area of expression of the proapoptotic p53 protein in "old" cultures of endotheliocytes by 2.3 times compared with the control. In the control "old" cultures of endotheliocytes, the expression area of Cx43 was 0.70±0.01%, which did not significantly differ from this indicator in "young cultures" (0.80±0.01)%. In "young" cultures of endotheliocytes, the expression area of Cx43 with the addition of the H-Asp(Ala)-OH peptide was 1.34±0.07%, which is 1.68 times more than in the corresponding control (Fig. 2).

Data on the effect of the H-Asp(Ala)-OH peptide on the expression of Ki67, p53, and Cx43 proteins in cultures of human endotheliocytes during their aging may play an important role in understanding the molecular mechanisms of the antiatherosclerotic action of this peptide. It is known that the violation of the integrity of the endothelial lining of the intima of arteries can be caused by apoptotic death of vascular endothelial cells. The reason for the development of apoptosis of vascular endothelial cells in atherosclerosis may be the induction of lipid peroxidation and the accumulation of active oxygen radicals. A study conducted on coronary arteries taken from 52 deceased patients with atherosclerosis and coronary artery disease at various stages of atherogenesis showed an increase in the apoptotic index of endotheliocytes in coronary arteries affected by atherosclerosis compared to undamaged segments of the vessels of the control group. At the same time, the processes of apoptosis of vascular endothelial cells proceed intensively only in the early stages of the atherosclerotic process [Vladimirskaya T.E., Shved I.A., Krivorot S.G. Apoptosis of coronary artery wall cells as a factor in the development and progression of coronary sclerosis. Topical issues of cardiology. 2013. No. 9. P. 22–26]. Expression of Cx43 is sharply reduced at sites of atherosclerotic vascular lesions in humans and animals. Atherosclerotic lesions of the human aorta are characterized by a sharp decrease in the number of gap junctions, which is determined by a decrease in the expression of Cx43. That is, with the development of atherosclerotic lesions, the three-dimensional cellular network formed by gap junctions is destroyed [Orekhov A.N., Andreeva E.R., Bobryshev Y.V. Cellular mechanisms of human atherosclerosis: Role of cell-to-cell communications in subendothelial cell functions // Tissue Cell. 2016. Vol. 48. N 1. P. 25-34]. Therefore, stimulation of proliferation (expression of the Ki67 protein), formation of intercellular contacts (expression of Cx43), and reduction of apoptosis (p53 protein) under the action of the H-Asp(Ala)-OH peptide may indicate the ability of this dipeptide to prevent the development of atherosclerosis at its initial stage.

Example 5 . Effect of H-Asp(Ala)-OH Peptide on Endothelin-1 Expression in Human Vascular Endothelium in Atherosclerosis

Four tissue fragments of human atherosclerotic aorta with a diameter of 0.2 cm were obtained during coronary artery bypass grafting. The tissue was crushed, poured with 10 ml of HBSS supplemented with 0.2% collagenase I (Gibco), centrifuged at 400 g, and incubated for 20 min at 37°C in a thermostat. After centrifugation, the cell pellet was dissolved in 2 ml of M199 medium supplemented with 10% fetal bovine serum and an antibiotic. The isolation cycle was repeated 3 times, the dissolved sediment of endotheliocytes was accumulated in a separate tube. The cell concentration for seeding was 3x10 ⁴ cells per ml. Cells were grown to passage 3 ("young" culture) and passage 6 ("old" culture). Since the endothelium was isolated from the tissue of vessels affected by atherosclerosis, cell proliferation in culture was observed only up to the 6th passage, after which most of the endotheliocytes entered into apoptosis. At passages 3 and 6, cells were divided into 2 groups: 1 - control (adding culture medium), 2 - adding H-Asp(Ala)-OH peptide at a concentration of 10 ng/ml. The study of endothelin-1 expression was carried out by immunofluorescent method using primary antibodies to anti-endothelin-1 (monoclonal, 1:100, Novocastra). Antibodies conjugated with Alexa Fluor 647 fluorochrome (1:1000, Abcam) were used as secondary antibodies. Cell nuclei were counterstained with Hoechst 33258 (Sigma, USA) for 1 min.

Endothelin-1 is a peptide synthesized by the vascular endothelium, which has vasoconstrictor and mitogenic properties. Endothelin-1 has a wide range of biological effects: it activates the proliferation of fibroblasts, the synthesis of extracellular matrix components, vasoactive substances (prostacyclin, nitric oxide), platelet activating factors. Violation of the synthesis of endothelin-1 is a sign of endothelial dysfunction and is observed in the pathology of the vessels of the retina, kidneys, heart, lungs and other organs.

The results were analyzed using an Olympus FluoView 1000 confocal microscope (Japan) and Olympus FluoView ver 3.1b software. In each case, 10 fields of view were analyzed at a magnification of 200. The expression of endothelin-1 was assessed by the expression brightness per cell in arbitrary units (c.u.). Statistical processing of the experimental data included the calculation of the arithmetic mean, standard deviation, and confidence interval for each sample and was carried out using the Statistica 6.0 program. To analyze the type of distribution, the Shapiro-Wilk test was used. The critical confidence level of the null hypothesis was taken equal to 0.05.

In the "young" and "old" control cultures of endotheliocytes, the expression area of endothelin-1 did not differ and amounted to 13.8±0.2 c.u. Peptide H-Asp(Ala)-OH did not affect the expression of endothelin-1 in "young" endothelial cultures, where this indicator was 13.5±0.2 c.u. and increased this indicator in the "old" cultures by 2 times to 28.9±0.1 c.u.

Thus, the H-Asp(Ala)-OH peptide stimulates the synthesis of endothelin-1 in human endotheliocytes in atherosclerosis in a model of replicative cellular aging. This effect indicates the ability of the peptide to increase the functional activity of the endothelium in atherosclerosis.

Example 6 . Effect of a pharmaceutical composition based on the H-Asp(Ala)-OH peptide on vascular function in patients with cerebrovascular disease

A clinical study was conducted in 152 patients with vascular atherosclerosis in the form of cerebrovascular disease aged 62 to 78 years, who were divided into 4 groups by simple randomization. Patients in the control group (n=20) received conventional treatment - actovegin 200 mg 2 times a day for 30 days. Patients of the main group 1 (OG-1, n=43), in addition to conventional treatment, received a pharmaceutical composition based on the peptide β-aspartyl-alanine of the formula H-Asp(Ala)-OH at doses of 10.0 μg/kg (OG-1/ 1, n=24) or 100.0 μg of the peptide per 1 kg of body weight (OG-1/2, n=19) orally 1 capsule 2 times a day daily for 30 days. The second main group (OG-2) included 48 people who received a pharmaceutical composition based on the dipeptide β-aspartyl-alanine of the formula H-Asp(Ala)-OH in the form of a solution intranasally, 5 drops in each nasal passage at doses of 1.0 mcg/kg (OG-2/1, n=26) or 10.0 mcg/kg body weight (OG-2/1, n=22) per day for 30 days. The third main group (OG-3) included 41 people who received a pharmaceutical composition based on the dipeptide β-aspartyl-alanine of the formula H-Asp(Ala)-OH in the form of a solution for intramuscular use, 1 ml at doses of 0.1 μg / kg of body weight (OG-3/1, n=20) or 10.0 µg/kg of body weight (OG-3/2, n=21) per day for 30 days. Patients complained of frequent headaches, dizziness, emotional lability, reduced mental performance. A laboratory study revealed a violation of lipid metabolism, which was expressed in a significant increase in serum total cholesterol up to 7.9 ± 0.5 mmol / l, compared to the norm, low-density lipoproteins (LDL) up to 3.78 ± 0.06 mmol / l, triglycerides up to 2.7 ± 0.09 mmol/l, which correlated with the clinical manifestations of the disease (Table).

After the use of conventional means in patients of all groups, subjective and laboratory parameters improved compared with those before treatment. However, in patients of the main groups, the positive dynamics was significantly more pronounced. In particular, the content of total cholesterol decreased to 5.9±0.6 mmol/l in OG-1, 5.7±0.4 mmol/l in OG-2 and 5.6±0.7 mmol/l in OG-3, approaching the upper limit of the norm (p<0.05 compared with the index before treatment and with the index in patients after treatment with conventional means), while in the control group the index decreased only to 7.0 ± 0.6 mmol/ l. Similar dynamics was observed in LDL and triglycerides: in patients of the main groups after the course of treatment, the indicators approached the physiological norm, while in patients of the control group who received only conventional treatment, these indicators improved relative to the initial values, but remained outside the normal range . The dynamics of subjective indicators correlated with the dynamics of laboratory parameters. So, in 75-83% of patients of the three main groups, the frequency of headaches, sleep disturbances decreased, mental performance increased, and manifestations of emotional lability decreased. Positive dynamics of subjective indicators was noted in 60% of patients in the control group. Thus, the results of the clinical study indicate that the effectiveness of the treatment of patients with arterial atherosclerosis in the form of cerebrovascular disease significantly increases when a pharmaceutical composition based on the H-Asp(Ala)-OH peptide is included in the complex therapy compared with patients who received only the generally accepted treatment.

Table - The effect of a pharmaceutical composition based on the peptide H-Asp(Ala)-OH on lipid metabolism in patients with arterial atherosclerosis

Indicator Norm Before treatment After treatment with conventional means
(the control) After treatment with
H-Asp(Ala)-OH OG-1 OG-2 OG-3 total cholesterol,
(mmol/l) 3.2–5.6 7.9±0.5 7.0±0.6* OG-1/1
5.9±0.6*^
OG-1/2
5.7±0.3*^ OG-2/1
5.7±0.4*^
OG-2/2
5.6±0.5*^ OG-3/1
5.6±0.7*^
OG-3/2
5.4±0.5*^ Low density lipoproteins (LDL), (mmol/l) 1.71–3.5 3.78±0.06 3.44±0.08* OG-1/1
3.21±0.02*^
OG-1/2
3.16±0.07*^ OG-2/1
3.16±0.03*^
OG-2/2
3.12±0.08*^ OG-3/1
3.08±0.04*^
OG-3/2
3.03±0.08*^ Triglycerides, (mmol/l) 0.41–1.8 2.7±0.09 2.3±0.07* OG-1/1
2.1±0.08*^
OG-1/2
2.0±0.06*^ OG-2/1
2.0±0.09*^
OG-2/2
1.8±0.05*^ OG-3/1
1.9±0.1*^
OG-3/2
1.6±0.06*^

* р<0.05 – significantly compared with the index before treatment;

^ p<0.05 - significant compared with the indicator in the control group.

Claims (3)

1. The use of the β-aspartyl-alanine peptide of the formula H-Asp(Ala)-OH to improve the functional activity of the vascular endothelium. 2. The use of the β-aspartyl-alanine peptide of the formula H-Asp(Ala)-OH for the preparation of a drug that improves the functional activity of the vascular endothelium. 3. A method for the prevention and treatment of endothelial dysfunction in organs and tissues, which consists in administering to the patient a drug that improves the functional activity of the vascular endothelium, including the β-aspartyl-alanine peptide of the formula H-Asp(Ala)-OH, while parenteral administration of the drug dose means is 0.1-10 mcg / kg body weight at least once a day, with intranasal - 1.0-10 mcg / kg body weight at least once a day, with oral - 10.0-100 mcg /kg of body weight at least twice a day for the period necessary to achieve a therapeutic effect.

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