RU2667466C1 - Peptide compound, restoring function of respiratory bodies and inflammable immunomodulating action - Google Patents

Abstract

FIELD: medicine; pharmaceuticals.SUBSTANCE: group of inventions relates to medicine and concerns the tetrapeptide alanyl-glutamyl-aspartyl-leucine amide of the general formula Ala-Glu-Asp-Leu-NH. Group of inventions also relates to the use of said tetrapeptide for restoring respiratory function and immunocorrection; pharmacological agent of a peptide nature restoring respiratory function and having an immunomodulatory effect, comprising an active peptide agent and a pharmaceutically acceptable carrier.EFFECT: group of inventions provides a complex restorative effect on the function of the respiratory system, including immunocorrection.3 cl, 6 dwg, 9 tbl, 9 ex

Description

The invention relates to medicine, in particular to medicines, and can be used as a means of restoring the function of the respiratory system and at the same time having an immunomodulating effect.

Currently, there is an upward trend in respiratory diseases, in particular non-specific lung diseases, which occupy the third place in the world in prevalence, among which chronic obstructive pulmonary disease (COPD) is an extremely common disease, which is one of the main causes of disability, disability, significantly reducing the quality of life of patients and taking the fourth place among the causes of death in industrialized countries. Moreover, the relevance of this problem is increasing every day: if over the past decade the overall mortality and mortality from cardiovascular diseases has been decreasing, then mortality from COPD has increased by 28% (Aisanov Z.R., Kokosov A.N., Ovcharenko S.I. , Khmelkova N.G., Tsoi A.N., Chuchalin A.G., Shmelev E.I. Chronic obstructive pulmonary diseases.Federal program // Breast Cancer; 2001; 1: 9–33; Respiratory Diseases: A Manual for Doctors : in 4 t. Under the general editorship of NR Paleev. T.1. General pulmonology. M: Medicine, 1989). It is difficult to accurately determine the prevalence of COPD due to terminological uncertainty that has existed for many years. According to some studies, this indicator ranges from 10% to 30%. In the United States, COPD affects about 14 million people, in the UK - 900 thousand people, in Russia - 11 million (Dvoretsky L.I. Infection and chronic obstructive pulmonary disease // Consilium Medicum. 2001; 3 (12): 587- 595). According to the World Health Organization, there is a trend towards a steady increase in mortality from this type of pathology (NHLBI / WHO Workshop Summary: Global strategy for the diagnosis, management and prevention of Chronic Obstructive Pulmonary Disease. Am. J. Respir. Crit. Care Med. 2001; Vol. 163: 1256-1270; chronic obstructive pulmonary disease / Edited by A. G. Chuchalin. - M.: Ministry of Health, Russian Federation Research Institute of Pulmonology, Ministry of Health of the Russian Federation, 1998).

The term COPD appeared about 30 years ago and combines diseases characterized by a slowly but steadily progressing irreversible bronchial obstruction with increasing symptoms of chronic respiratory failure. The COPD group includes chronic obstructive bronchitis, pulmonary emphysema, severe bronchial asthma, and cystic fibrosis obliterating bronchiolitis and bronchiectasis in the USA and Great Britain (Diseases of the respiratory system: Manual for doctors: in 4 t. Under the general editorship of N.R. Paleev T. 1. General pulmonology.M .: Medicine, 1989).

The treatment of this pathology is a difficult task. With exacerbation of COPD, the leading etiological factor is infectious. The main bacterial pathogens are H. influenzae, M. catarrhalis (much more often in the winter season), S. Pneumoniae, Str. Aureus, as well as Enterobactericae and P. aeruginosa. It is important to remember the role of viruses in infectious exacerbations of COPD (up to 30% of cases), among which rhinoviruses prevail, and influenza A and B viruses are detected much less frequently (Seemungal T, Donaldson GC, Breuer J, Jhonston IS, Jeffries DJ, Wedzicha JA. Rinoviruses are associated with exacerbations of COPD. Eur Resp J. 1998; 12 (Suppl. 28): 298).

A comprehensive approach is needed in the treatment of COPD. Of the drugs, bronchodilators constitute the basic therapy, since it is bronchial obstruction that plays a paramount role in the pathogenesis of COPD Barnes PJ. Bronchodilators: basic pharmacology. In Calverley P, Pride N, eds. Chronic obstructive pulmonary disease. London: Chapman and Hall, 1995; 391-417). Although irreversible bronchial obstruction occurs in COPD, the use of bronchodilators can reduce the severity of shortness of breath and other symptoms of COPD in approximately 40% of patients and increase exercise tolerance. In accordance with the GOLD recommendations, the choice of a particular group of bronchodilators (M-anticholinergics, b2-agonists and methylxanthines) and their combinations is made individually for each individual patient, depending on the severity of the disease and the characteristics of its progression, the nature of the response to treatment and the risk of side effects as well as the availability of medicines. If the maximum doses of bronchodilators are ineffective, glucocorticosteroid therapy is used, which improves bronchial patency in 10-30% of patients and requires trial treatment before prescribing for long-term use.

Antibacterial therapy is carried out exclusively during an exacerbation of COPD. Recently, annual preventive vaccination of all patients with COPD with influenza vaccine has come to the forefront, reducing the mortality rate of patients by about 50%, which can reduce the number of exacerbations of the disease, their duration and severity, and therefore, improve bronchial patency, reduce the number of days of disability and improve the quality of life of patients (Tsoi A.N., Arkhipov V.V. Evidence-based pharmacotherapy of chronic obstructive pulmonary disease // Consilium Medicum. 2002; 04 (09): 486-492.; Butler L I.I. Infection and chronic obstructive pulmonary disease // Consilium Medicum. 2001; 3 (12): 587-595).

Mucolytic agents are also widely used for the treatment of COPD, the main therapeutic effect of which is to directly dilute the pathologically viscous secretion by changing the composition and amount of mucus glycoprotein, which is secreted by the cells of the epithelial lining of the respiratory tract. The goal of mucolytic therapy is to reduce coughing and facilitate sputum discharge.

A known preparation of a polypeptide nature (RF patent No. 1681426 for the invention "A method for producing a means of restoring the respiratory function of the lungs", 1989, IPC A 61K35 / 24), obtained from organic raw materials (animal trachea).

Known peptide compound that restores the function of the respiratory system (patent RU2255757, IPC A61K 38/00, publ. 07/10/2005), which is an alanyl-glutamyl-aspartyl-leucine tetrapeptide of the general formula Ala-Glu-Asp-Leu [SEQ ID NO : 1], with biological activity, manifested in the restoration of respiratory function. Also known is a peptidic pharmacological agent that restores respiratory function, contains an active peptide agent and a pharmaceutically acceptable carrier, an effective amount of Ala-Glu-Asp-Leu tetrapeptide is used as the active peptide agent [SEQ ID NO: 1]. The known compound has similar biological activity and is the closest analogue in chemical structure to the claimed peptide (it should be noted that the claimed new peptide compound - the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ has no structural analogues).

The possibility of using the known compound as the basis of drugs for the treatment and prevention of a number of pulmonological diseases and the restoration of respiratory function is limited by a number of its disadvantages: rapid biodegradation of the prototype in the blood and body tissues; poor bioavailability of the prototype when taken orally; low biological activity of the prototype. In addition, the known compound does not have pronounced immunomodulatory properties.

The objective of the invention is to obtain a new biologically active compound of peptide nature, effectively restoring the function of the respiratory system and having immunomodulating properties.

The technical result of the claimed invention is a comprehensive restorative effect on the function of the respiratory system, including immunomodulation.

The technical result is achieved by the tetrapeptide alanyl-glutamyl-aspartyl-leucine amide of the general formula Ala-Glu-Asp-Leu-NH ₂ .

The technical result is also achieved by the use of the tetrapeptide alanyl-glutamyl-aspartyl-leucine amide of the general formula Ala-Glu-Asp-Leu-NH ₂ to restore respiratory function and immunocorrection.

The technical result is also achieved by a pharmacological agent of a peptide nature, restoring the function of the respiratory system and having an immunomodulating effect, containing an active peptide agent and a pharmaceutically acceptable carrier. In contrast to the prototype, the active peptide agent contains an effective amount of the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ in a dose of 0.01-1000 μg / kg body weight.

The possibility of an objective manifestation of a technical result when using the invention is confirmed by reliable data given in the examples containing experimental information obtained by the methods adopted in this field.

The invention is illustrated by illustrations.

FIG. 1. The structural formula of the claimed tetrapeptide H-Ala-Glu-Asp-Leu-NH ₂ ;

FIG. 2. Scheme for the synthesis of the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ in solution using the mixed anhydride method;

Figure 3. Kinetics of tetrapeptide biodegradation [³ H] Ala-Glu-Asp-Leu-NH₂and [³ H] Ala-Glu-Asp-Leu in rat plasma treated with heparin;

Figure 4. Pharmacokinetic curves of Ala-Glu-Asp-Leu-NH tetrapeptides₂and Ala-Glu-Asp-Leu in rat plasma after intravenous administration of tetrapeptides at a dose of 15 mg / kg;

Figure 5. Pharmacokinetic curves of Ala-Glu-Asp-Leu-NH tetrapeptides₂and Ala-Glu-Asp-Leu in rat plasma after oral administration of tetrapeptides at a dose of 50 mg / kg;

6. Schematic diagram of an experimental setup for modeling COPD by fumigating experimental animals with cigarette smoke. Figure 6 shows: 1 - exhaust pipe; 2 - Cabin for animals; 3 - Switchgear; 4 - Clean air; 5- Suction pump; 6 - Valve No. 1; 7 - Valve No. 2; 8 - a cigarette; 9 - Electronic unit.

The tetrapeptide Ala-Glu-Asp-Leu-NH ₂ can be obtained by any known method, both using various methods of synthesis in solution, and using various methods of solid-phase synthesis. An example is the preparation of this tetrapeptide in solution using the mixed anhydride method (FIG. 2).

1. Name of compound: L-alanyl-L-glutamyl-L-aspartyl-L-leucine amide

2. The structural formula of the tetrapeptide H-Ala-Glu-Asp-Leu-NH ₂ shown in figure 1.

3. Gross formula without counterion: C ₁₈ H ₃₁ N ₅ O ₈

4. Molecular weight without counterion: 445.48

The following abbreviations are used in the description of the synthesis:

Ala-Glu-Asp-Leu-NH ₂ - L-Alanyl-L-Glutamyl-L-Aspartyl-L-Leucine Amide

Boc-Glu (OBzl) -OH x DCCA-dicyclohexylamine salt of N-carbotretbutyloxy-γ- (O-benzyl) glutamic acid;

Boc-Glu (OBzl) -OH- N-carbotretbutyloxy-γ- (O-benzyl) -glutamic acid;

Boc-Asp (OBzl) -OH - N-carboxytet-butyloxy-β- (O-benzyl) aspartic acid;

Leu-NH ₂ xHCl - leucine amide hydrochloride;

Boc-Asp (OBzl) -Leu-NH ₂ -N-carboxytetbutyloxy-β- (O-benzyl) aspartyl-leucine amide;

Asp (OBzl) -Leu-NH ₂ * CF ₃ COOH - β- (O-benzyl) aspartyl-leucine amide trifluoroacetate;

Boc-Glu (OBzl) -Asp (OBzl) -Leu-NH ₂ —N-carbotretbutyloxy-γ- (O-benzyl) -glutamyl-β- (O-benzyl) -partyl-leucine amide;

Glu (OBzl) -Asp (OBzl) -Leu-NH ₂ * CF ₃ COOH - γ- (O-benzyl) -glutamyl-β- (O-benzyl) -partyl-leucine amide trifluoroacetate;

Z-Ala-OH-carbobenzoxy-alanine;

Z-Ala-Glu (OBzl) -Asp (OBzl) -Leu-NH ₂ - carbobenzoxy-alanyl-γ- (O-benzyl) -glutamyl-β- (O-benzyl) -partyl-leucine amide;
NMM is N-methylmorpholine;

TFA-trifluoroacetic acid;

Pd / C - palladium on carbon;

DCC - dicyclohexylcarbodiimide;

HOBt-hydroxybenzotriazole;

IBCF-isobutylchloroformate;

DCCA-dicyclohexylamine;

DMF-dimethylformamide;

IPA isopropyl alcohol.

Synthesis of Boc-Asp (OBzl) -Leu-NH ₂ .

Dissolve 2.16 g (0.00667 Mol) Boc-Asp (OBzl) -OH in 10.0 ml of DMF, cooled to ^-25 ° C and add 0.89 ml (0.935 g, 0.00669 moles) of 98% IBCF . After achieving a temperature ^of -25 C start dropwise with stirring over 15-20 min 0.74 ml (0,0,68 g, 0,00682Mol) NMM, maintaining the temperature not higher than -20 ° ^C. We maintain the reaction mixture 20 minutes at -20 ^{° C,} then cooled to -40 ° ^C whereupon 1.14 g (0.0068 Mol) Leu-NH ₂ xHCl, suspended in 7.5 ml of DMF, cooled to -10 ° ^C. Then add 0.82 ml (0.75 g, 0.734 mol) of NMM. Cool the reaction mixture to -20 ^° C maintained at this temperature for 90 minutes. Slowly raise the temperature of the reaction mass to room temperature. Then leave the reaction mass overnight. We evaporate on a rotary evaporator with a residual pressure of not higher than 3 mm Hg. and the temperature in the bath is not higher than +42 ^о С until the end of solvent distillation. The residue is dissolved in 30 ml of ethyl acetate at room temperature (about +20 ^about C). The resulting solution at room temperature is washed with 7.5 ml of water, 3% citric acid solution, and then two more times with water. We distill off ethyl acetate on a rotary evaporator at a residual pressure of no higher than 40 mm Hg. and the temperature in the bath is not higher than +42 ^о С until the end of solvent distillation. The residue is overwritten in a mixture of hexane (15 ml) with diethyl ether (1.5 ml) at room temperature. Filter the precipitate, wash it twice with 7.5 ml of hexane at room temperature. The resulting precipitate dry them in a convection oven at a temperature not higher than 40 ^{° C} to constant weight. The yield of Boc-Asp (OBzl) -Leu-NH ₂ was 2.32 g (96%).

Synthesis of Boc-Glu (OBzl) -Asp (OBzl) -Leu-NH ₂

To 1.87 g (0,0043Mol) Boc-Glu (OBzl) -Asp (OBzl) -Leu-NH ₂ under cooling in an ice bath add 6.75 ml cooled to 5 ^{° C} with trifluoroacetic acid. Stir until dissolved, remove the ice bath, incubate an additional 30 minutes. We evaporate on a rotary evaporator with a residual pressure of not more than 20 mm Hg. and the temperature in the bath is not higher than +40 ^о С until the end of trifluoroacetic acid distillation, we evaporate 2 times 7.5 ml of benzene under the same conditions. The residue was dissolved in 9 ml of DMF, cool the resulting solution to -12 ° ^C. Then add NMM (0.4-0,7 mL) until pH = 7.3-7.5. Adding NMM is carried out immediately before adding this solution to a solution of mixed anhydride from Z-Glu (OBzl) -OH and IBCF.

To the cooled +10 to + 5 ^{° C} slurry Boc-Glu (OBzl) -OH DCHA x 2.47 g (0.00476 mol) in 30 ml of ethyl acetate were added 12 ml cooled to 10 + 5 ^C a 20% solution citric acid (24 g of citric acid monohydrate) to pH = 4-5, mix the resulting mixture for 5 minutes, separate the organic layer, acidify the aqueous layer with a 30% solution of sulfuric acid at room temperature to pH = 3 and extract twice with 7.5 ml of ethyl acetate at room temperature. We wash the combined extract twice with 4.5 ml of 3% solution of citric acid and twice with 4.5 ml of water (water temperature and room citric acid solution). Evaporate ethyl acetate on a rotary evaporator at a residual pressure of not higher than 40 mm Hg. and the temperature in the bath is not higher than +40 ^о С until the end of solvent distillation. The residue is evaporated with 7.5 ml of DMF to remove water on a rotary evaporator at a residual pressure of no higher than 3 mm Hg. and the temperature in the bath is not higher than +40 ^о С until the end of solvent distillation. The weight of the obtained oil was 1.92 g. Product yield 95% (0.00377 mol, 1.52 g Boc-Glu (OBzl) -OH).

Dissolve the resulting oil 1.92 g (0.00452 Mol, 1.52 g of Boc-Glu (OBzl) -OH) in 9 ml of DMF, cooled to - 30 ^{° C} and added in one portion 0.59 mL (6.17 g, 0 , 00452 mol) 98% IBCF. The temperature of the reaction mass is kept at between - 18 ^C to - 20 ° ^C was kept for 20 minutes at - 20 ° ^C is cooled to a temperature of - 35 ^C to - 40 ^C. Then add a solution of Asp (OBzl) - Leu-NH _2, prepared from 1.87 g (0.0043 Mol) Boc-Asp (OBzl) -Leu -NH _2, and dissolved in 9 ml of DMF, cooled to ^-20 ° C, to which was then added 0.4- 0.7 ml NMM, until a pH of 7.3-7.5 is reached. The temperature rises to -15 ° ^C. The reaction mixture was recooled to -20 ° ^C. At this temperature, stirred for 90 min. Slowly raise the temperature to room temperature and leave overnight. We evaporate on a rotary evaporator with a residual pressure of not higher than 3 mm Hg. and bath temperature not exceeding + 40 ° ^C. The residue is dissolved in 2.4 ml ethyl acetate at room temperature. Then we wash the ethyl acetate solution in 4.5 ml: water, 3% citric acid solution two more times with water. The temperature of the water and citric acid solution is room temperature. We distill off ethyl acetate on a rotary evaporator at a residual pressure of no higher than 40 mm Hg. and bath temperature not exceeding + ⁴⁰ ° C to distill off the solvent closure. The residue was dissolved in 30 ml of IPA at a temperature of + 65-75 ^{° C} and the resulting solution was subject to crystallization at room temperature. We filter the precipitate, wash it on the filter two times with 7.5 ml of isopropyl alcohol at room temperature, twice with 7.5 ml of hexane at room temperature. Dry the washed precipitate in conventional oven at a temperature not higher than + 40 ^{° C} to constant weight. The yield of the Boc-Glu (OBzl) -Asp (OBzl) -Leu-NH ₂ compound was 2.54 g (90%).

Synthesis of Z-Ala-Glu (OBzl) -Asp (OBzl) -Leu-NH ₂ .

To a 2.828 g (0,0043Mol) Boc-Glu (OBzl) -Asp (OBzl) -Leu -NH ₂ under cooling with ice bath, add 5.4 mL, cooled to +5 ^C. TFA. Stir until dissolved, remove the ice bath. We stand for another 30 minutes. We evaporate the resulting solution on a rotary evaporator at a residual pressure of not more than 20 mm Hg. and the temperature in the bath is not higher than + 40 ^о С until the end of the distillation of TFU. We evaporate twice 7.5 ml of benzene under the same conditions. The residue was dissolved in 9 ml of DMF, cool the resulting solution to a temperature of - 12-15 ^C, add NMM (0,4-0,6 ml) until pH = 7.3-7.5. NMM addition to this solution is carried out immediately before the addition of a mixed anhydride solution from Z-Ala-OH and IBCF to this solution.

Dissolve 1.009 g (0.00452 mol) of Z-Ala-OH in 7.2 ml of DMF. Cooled to - 30 ^° C and in one portion to this solution was added with stirring 0.59 ml (0.62 g, 0.00452 moles) of 98% IBCF. Then with stirring, add 0.49 ml (0.46 g, 0, 00452 mol) of NMM. The temperature when added support from - 18 ^C to - 20 ° ^C. We maintain the reaction at a temperature of seven - 20 ^{° C} for 20 min. The solution is cooled to a temperature of - 35 ^C to - 40 ^C. To the reaction mixture add Glu (OBzl) solution -Asp (OBzl) -Leu-NH ₂ obtained from 2.828 g (0.0043 Mol) Glu (OBzl) -Asp (OBzl) -Leu-NH ₂ dissolved in DMF and cooled to - ²⁰ ° C, to which is then added 0.4-0.6 ml NMM to achieve a pH = 7.3-7.5. The temperature is raised to - 15 ^C. The reaction mixture was cooled to - 20 ^° C maintained at that temperature with stirring for 90 minutes. Then slowly raise the temperature to room temperature and leave overnight.

We evaporate on a rotary evaporator with a residual pressure of not higher than 3 mm Hg. and bath temperature not exceeding + ⁴⁰ ° C to completely remove the solvent. The residue is dissolved in 24 ml of ethyl acetate at room temperature. The resulting solution is washed with 4.5 ml: water, 3% citric acid solution, water twice, 3% citric acid solution. We distill off ethyl acetate on a rotary evaporator at a residual pressure of no higher than 40 mm Hg. and bath temperature not exceeding + ⁴⁰ ° C to distill off the solvent closure. The residue was dissolved in 30 ml of IPA at a temperature of + 65-75 ^{° C} and left to crystallize at room temperature. The precipitate is filtered, washed twice with 7.5 ml of IPA at room temperature, twice with 7.5 ml of hexane at room temperature. The washed precipitate dry them in a convection oven at a temperature and + 40 ^{° C} to constant weight. The yield of Z-Ala-Glu (OBzl) -Asp (OBzl) -Leu-NH ₂ compound was 2.72 g (83%).

Synthesis of Ala-Glu-Asp-Leu-NH ₂ .

Dissolve 7.5 g (0.00987 Mol) Z-Ala-Glu (OBzl ) -Asp (OBzl) -Leu-NH ₂ in a minimum quantity of DMF (approximately 18 ml) at + 40 ° ^C. To the resulting solution add 18 ml of acetic acid. To the resulting solution we add 1.2 g of 5% palladium on carbon (Sigma-Aldrich 205680 SPEC) suspended in 4.5 ml of water, and immediately begin to pass an intense stream of hydrogen gas. Hydrogenated reaction mass at a temperature of + 40-42 ^° C during the hydrogenation reaction can be released from the sludge mass. Therefore, first (1-2 hours after the start of the hydrogenation process), we add 7.5-15 ml of a 1: 1 mixture of acetic acid and DMF, and then after 10-15 hours we add 7.5-9 ml of water after repeated precipitation. The hydrogenation process is performed within 18-20 hours. After the end of hydrogenation, we filter out palladium on charcoal. Coal is washed two or three times with acetic acid, and then two or three times with warm water (with a temperature of + 40-50 ° ^C).

Evaporate the filtrate and washings separately. The filtrate and the combined washes are evaporated on a rotary evaporator at a temperature of no higher than +42 ^о С at a residual pressure of 20 mm Hg at the beginning. and then at a residual pressure of 5 mm Hg. Evaporation is carried out approximately 2/3 of the initial solution. After that, the still bottoms formed from evaporation of the filtrate and washing are combined together. To the resulting bottom residue, add 30 ml of IPA at room temperature and mix thoroughly for 2-5 minutes until crystals form. The resulting crystals are quickly filtered under a film. The crystals on the filter are washed three times with 15 ml of IPA at room temperature, then washed twice with 15 ml of hexane at room temperature and dried under vacuum on a rotary evaporator at a residual pressure of 20 mm Hg. and the temperature in the bath is not higher than + 40 ^о С to constant weight. The yield of Ala-Glu-Asp-Leu-NH ₂ compound was 4.17 g (95%).

To clarify the invention are examples.

Example 1. The study of the general toxic effect of the tetrapeptide Ala-Glu-Asp-Leu-NH _2.

The general toxic effect of the tetrapeptide Ala-Glu-Asp-Leu-NH _{2 was} studied in accordance with the requirements of the Guidelines for the experimental (preclinical) study of new pharmacological substances ”/ Edited by Corresponding Member, Professor R.U. Khabrieva - 2nd ed., Revised. and add. - M.: Publishing House "Medicine", 2005: 823.

The study of the general toxic effect of the tetrapeptide Ala-Glu-Asp-Leu-NH _{2 was} carried out in two stages: a) the study of "acute toxicity"; b) the study of "chronic" toxicity.

These studies were performed on SHR mice of both sexes aged 2-2.5 months and weighing 18-22 g, and on Wistar rats of both sexes aged 2-4 months and weighing 150-240 g.

With a single intramuscular and intragastric administration of the tetrapeptide to SHR mice of both sexes (35-140 μg / mouse) and Wistar rats of both sexes (350-1400 μg / rat), the toxic effect of the drug was not detected. The calculation of the toxic dose showed that for this tetrapeptide, the LD ₅₀ value is 1587 mg / kg for a single intragastric administration and 1111 mg / kg for a multiple intragastric administration for 24 days daily. The cumulation calculation was 0.70. This indicates a lack of addiction to this tetrapeptide. It should be noted that LD ₅₀ exceeds the estimated therapeutic dose for oral administration by 225,000 times.

Example 2. The study of biotransformation and lifetime of tetrapeptides Ala-Glu-Asp-Leu and Ala-Glu-Asp-Leu-NH ₂ in rat plasma.

To study the biotransformation of tetrapeptides Ala-Glu-Asp-Leu and Ala-Glu-Asp-Leu-NH ₂ , HPLC of the products of [ ³ H] Ala-Glu-Asp-Leu and [ ³ H] Ala-Glu-Asp-Leu- NH ₂ with rat blood plasma pretreated with heparin. Tritium-labeled these tetrapeptides with molar radioactivity of 55 and 65 Ci / mmol were used. When performing HPLC, a UV detector and a flow detector of radioactivity were used in series. Chromatography was performed on a column filled with a sorbent with a reversed phase C ₁₈ , particle size 5 μm (Luna 5u C18 (2) 100 A. A mixture of acetonitrile and a 0.05 M solution of monosubstituted potassium phosphate with a pH of 3 in a ratio of 1: 9 was used as the mobile phase .

To identify the peptide fragments that can be formed during biotransformation in the blood plasma of rats of these two [ ³ H] Ala-Glu-Asp-Leu and [ ³ H] Ala-Glu-Asp-Leu-NH ₂ tetrapeptides, 2-3 membered peptide fragments formed during various biodegradation pathways with the C and N ends of the peptide chain. To determine the kinetics of biodegradation, we used the initial concentration of these tetrapeptides of 7.0 μmol in rat heparin plasma.

The results of the studies showed that the hydrolysis of [ ³ H] Ala-Glu-Asp-Leu tetrapeptides and [ ³ H] Ala-Glu-Asp-Leu-NH ₂ tetrapeptides in rat heparin plasma under the action of peptidases occurred according to two different schemes. Hydrolysis of the tetrapeptide [ ³ H] Ala-Glu-Asp-Leu occurred under the action of diaminopeptidases present in rat plasma from the N-terminus of the peptide chain. At the same time, hydrolysis of [ ³ H] Ala-Glu-Asp-Leu-NH ₂ occurred under the action of dicarboxypeptidases from the C-terminus of the peptide chain. In addition, it was reliably shown that the tetrapeptide [ ³ H] Ala-Glu-Asp-Leu-NH ₂ , in comparison with the tetrapeptide [ ³ H] Ala-Glu-Asp-Leu, has a higher resistance to hydrolysis in rat heparin plasma .

The change in the concentration of tetrapeptides in rat heparin blood over time is shown in FIG. 3. The half-degradation period for the tetrapeptide [ ³ H] Ala-Glu-Asp-Leu-NH ₂ was 18 minutes. At that time, the same indicator for the tetrapeptide [ ³ H] Ala-Glu-Asp-Leu was 3 minutes.

Example 3. The study of the pharmacokinetics of tetrapeptides Ala-Glu-Asp-Leu and Ala-Glu-Asp-Leu-NH ₂ in the blood plasma of rats with oral and intravenous routes of administration.

To study the pharmacokinetics of Ala-Glu-Asp-Leu and Ala-Glu-Asp-Leu-NH ₂ tetrapeptides in rat blood plasma, a highly specific and highly sensitive method for their quantification was developed using high performance liquid chromatography in combination with a tandem mass spectrometric detector (API 3200 Q Trap, Applied Biosystems / MDS SCIEX, USA). The target compounds were detected by electrospray ionization (ESI) in the mode of registration of positive ions formed from these tetrapeptides. Chromatographic separation was carried out on a Zorbax Extend C18 column, 3.5 μm, 50 × 4.6 mm (Agilent, USA) in the gradient elution mode (flow rate 1 ml / min, total analysis time 3.5 min). The mobile phase consisted of acetonitrile and water containing 0.1% formic acid. Under these conditions, the retention times of Ala-Glu-Asp-Leu and Ala-Glu-Asp-Leu-NH ₂ tetrapeptides were 0.95 ± 0.01 min and 0.65 ± 0.01 min, respectively.

Extraction of tetrapeptides from rat plasma was carried out by solid phase extraction (TFE) in Oasis HLB, 30 μm (30 mg) extraction boards (Waters, Ireland). The limit of the quantitative determination (Lower Limit of Quantification - LLOQ) in the blood plasma of rat Ala-Glu-Asp-Leu and Ala-Glu-Asp-Leu-NH ₂ tetrapeptides was 70 ng / ml and 80 ng / ml, respectively, with a signal ratio / noise> 6. Calibration plots for these tetrapeptides had a linear relationship (r = 0.9976 and 0.9899 1 / x weighting factor) in the concentration range of 75 ÷ 10,000 ng tetrapeptide / ml plasma. These studies were performed on male Wistar rats of both sexes aged 2-4 months and weighing 150-240 g.

The study of the pharmacokinetics of these two tetrapeptides by oral and intravenous routes of administration was carried out on 20 Wistar male rats, divided into the following groups of equal number of animals:

Group number 1. Five rats were orally (using an intragastric tube) administered with Ala-Glu-Asp-Leu tetrapeptide at a dose of 50 mg / kg;

Group number 2. Five rats intravenously into the tail lateral vein of the tetrapeptide Ala-Glu-Asp-Leu at a dose of 5 mg / kg;

Group number 3. Five rats were administered orally (using an intragastric tube) with a dose of 50 mg / kg Ala-Glu-Asp-Leu-NH ₂ tetrapeptide;

Group number 4. Five rats intravenously into the tail lateral vein, the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ at a dose of 5 mg / kg;

In all four groups, sterile solutions of tetrapeptides were used for administration. Sterile saline was used to prepare these solutions.

The doses of each drug used were significantly higher than the estimated pharmacologically effective doses of tetrapeptides used to treat experimental pathological processes in rats. Blood samples were taken by decapitation of animals. Blood was collected in tubes containing EDTA 5, 10, 15, 20 and 30 minutes after oral and intravenous administration. Blood plasma was separated by centrifugation at 10,000 rpm for 15 min and immediately subjected to processing for HPLC-MS analysis.

In FIG. 4 graphically shows data on the change in the concentration of these investigated tetrapeptides in the blood of rats with the intravenous administration of these substances.

Figure 5 graphically shows the data on the change in the concentration of tetrapeptides in the blood of rats with the oral administration of these substances, the introduction of these substances.

The experimental data obtained on the change in the concentrations of these two tetrapeptides with oral and intravenous methods of administration were processed by mathematical statistics to obtain the following pharmacokinetic parameters:

AUC _{0 → ∞} (μg / ml x min) - the area under the graph "drug concentration - time", calculated from the time of administration to infinity ";

MRT (min) is the average residence time of a drug substance in the body;

To _el (min ^-1 ) is the elimination rate constant, a parameter that characterizes the rate of elimination of the drug from the body after oral or intravenous administration;

To _ab (min ^-1 ) is the absorption rate constant, a parameter that characterizes the rate of absorption of the drug into the body after oral administration;

V _ss (ml) is the stationary volume of the distribution of the substance;

Cl (ml / min) - total clearance - the amount of plasma that is purified from the drug per unit time;

T _1/2 (min) - the period for which half of the administered (or absorbed) dose of the drug substance is excreted;

The numerical values of these pharmacokinetic parameters are shown in table 1.

Table 1.

Pharmacokinetic parameters for tetrapeptides Ala-Glu-Asp-Leu and Ala-Glu-Asp-Leu-NH ₂ for oral and intravenous routes of administration

Parameter designation Ala-Glu-Asp-Leu Ala-Glu-Asp-Leu-NH ₂ Intravenous route of administration
Dose 15 mg / kg Oral route of administration
Dose 50 mg / kg Intravenous route of administration
Dose 15 mg / kg Oral route of administration
Dose 50 mg / kg AUC _{0 → ∞} , μg / ml x min 23.7 21.6 33.5 31,0 K _el , min -1 0.119 0.137 0,075 0,095 K _ab , min -1 - 0,089 - 0,099 V _ss , ml 35260 100603 35161 478688 T _1/2 min 5.79 5.03 8.17 10.47 Cl, ml / min 4214 4622 2981 3154

As can be seen from the data presented in table 1:

a) With the oral route of administration, a parameter such as K _ab for the new Ala-Glu-Asp-Leu-NH ₂ tetrapeptide is higher than for a similar parameter for the Ala-Glu-Asp-Leu tetrapeptide. This indicates that the new tetrapeptide Ala-Glu-Asp-Leu-NH ₂ compared with the prototype taken is better absorbed through the gastrointestinal tract, i.e. the amount of this new tetrapeptide that enters the systemic circulation is higher.

b) For oral and intravenous routes of administration, the parameter K _el for the new Ala-Glu-Asp-Leu-NH ₂ tetrapeptide is significantly lower than the specified parameter for the Ala-Glu-Asp-Leu tetrapeptide, i.e. this new tetrapeptide undergoes a markedly weaker biotransformation in the body.

c) With the oral and intravenous methods of administering Cl for the new Ala-Glu-Asp-Leu-NH ₂ tetrapeptide, it is significantly lower than the indicated parameter for the Ala-Glu-Asp-Leu tetrapeptide. This also indicates that this new tetrapeptide undergoes a markedly weaker biotransformation in the body.

Example 4. The study of the anti-inflammatory activity of tetrapeptides in a model of experimental bronchopneumonia caused by the introduction of turpentine in the bronchi of rats.

The study of the anti-inflammatory activity of tetrapeptides in a model of experimental bronchopneumonia caused by the administration of turpentine in rat bronchi was carried out on male Wistar rats aged 2-4 months and weighing 150-240 g.

Studies were performed on 60 male rats. All animals were divided into the following three groups, equal in number of animals (15 male rats in each group):

Group No. 1. (Intact group). Animals not exposed to turpentine, with additional administration of saline.

Group No. 2 (Control group). Animals with simulated acute bronchopulmonary inflammation with additional intraperitoneal administration of saline.

Group number 3. Animals with simulated acute bronchopulmonary inflammation followed by intraperitoneal administration of Ala-Glu-Asp-Leu tetrapeptide at a dose of 30 mg / kg;

Group number 4. Animals with simulated acute bronchopulmonary inflammation followed by intraperitoneal administration of the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ at a dose of 30 mg / kg

Acute bronchopulmonary inflammation (bronchopneumonia) in rats was modeled by the method described in detail in the work (Zarubina I.V., Mokrenko E.V., Bolekhan A.V., Shabanov P.D.Anti-inflammatory and immunomodulating activity of metaprot, trekrezan and polyoxidonium and their combinations in experimental bronchopulmonary inflammation in rats // Bulletin of the Smolensk State Medical Academy. 2016; 15 (1): 5-13).

Rats from group No. 1 were not exposed to any surgical exposure during the study.

Each rat from groups 2, 3, and 4 under ether anesthesia was surgically exposed to the trachea and an injection into the lumen between two cartilaginous half rings with a 0.8 mm diameter needle, 0.1 ml of turpentine was injected (according to GOST 1571-82). A cut on the neck was sutured. Then immediately after the operation and then every day for 5 days, animals from groups No. 1 and No. 2 were injected intraperitoneally with sterile saline; and the animals from group No. 3 and No. 4 were injected intraperitoneally with a sterile solution containing Ala-Glu-Asp-Leu and Ala-Glu-Asp-Leu-NH ₂ tetrapetides, respectively. On the fifth day of the study, the number of surviving animals was determined. Table 2 presents data on the number of surviving animals in each of the four groups on the 5th day of the study. Then the animals were decapitated and the lungs of the animals were separated. The lungs were stored in liquid nitrogen. After that, the tissue was crushed, homogenized, and biochemical parameters characterizing the energy metabolism of animal lung tissue cells were determined:

a) Lactic acid content (Lactate);

b) The content of pyruvate;

c) The content of adenosine triphosphoric, adenosine diphosphoric and adenosine monophosphoric acids;

g) the content of malondialdehyde;

d) The content of diene conjugates.

Table 2.

The effect of the introduction of tetrapeptides on the survival of rats in modeling acute bronchopulmonary inflammation.

Group number The number of animals taken for research, pieces The number of surviving animals on the 5th day of the study, pieces The share of surviving animals on the 5th day of research,% Group No. 1. (Intact group) fifteen fourteen 93.0 Group No. 2
(Control group. Bronchopulmonary inflammation) fifteen 5 30,0 Group No. 3 (Bronchopulmonary inflammation and administration of Ala-Glu-Asp-Leu) fifteen 9 60.0 Group No. 3 (Bronchopulmonary inflammation and administration of Ala-Glu-Asp-Leu-NH ₂ ) fifteen 12 80.0

As can be seen from the experimental data presented in Table 2, the administration of tetrapeptides to rats with acute bronchopulmonary inflammation significantly increases animal survival for 5 days. However, the new Ala-Glu-Asp-Leu-NH ₂ tetrapeptide more effectively enhances animal survival than the prototype taken for comparison.

Table 3.

The effect of tetrapeptides on metabolic processes in the lungs of rats in modeling acute bronchopulmonary inflammation.

Indicators Groups of animals Group No. 1. (Intact group) Group No. 2 (Control group)
Bronchopulmonary inflammation Group number 3
Bronchopulmonary inflammation and administration of Ala-Glu-Asp-Leu Group No. 4 Bronchopulmonary inflammation and administration of Ala-Glu-Asp-Leu-NH ₂ ) Lactate, μmol / g of tissue 3.06 ± 0.14 7.66 ± 0.12 5.17 ± 0.16 3.88 ± 0.11 * Pyruvate, μmol / g of tissue 0.29 ± 0.03 0.06 ± 0.03 0.16 ± 0.04 0.22 ± 0.05 * Lactate / pyruvate ratio 10.6 127.7 32.3 17.6 ATP, μmol / g of tissue 2.84 ± 0.08 1.35 ± 0.07 1.88 ± 0.06 * 2.55 ± 0.06 * ADP, μmol / g of tissue 0.75 ± 0.07 1.29 ± 0.06 0.95 ± 0.07 * 0.81 ± 0.05 * AMP, μmol / g of tissue 0.44 ± 0.05 0.87 ± 0.04 0.65 ± 0.08 * 0.51 ± 0.09 * Energy charge, conv. units 0.798 0.568 0.677 0.809 Malondialdehyde, nMol / g of tissue 4.52 ± 0.35 9.14 ± 0.41 7.52 ± 0.52 5.92 ± 0.48 * Diene conjugates, nmol / g of tissue 27.1 ± 2.8 57.6 ± 4.9 38.8 ± 5.2 31.1 ± 3.8 *

* Note. * p <0.05 compared with Group No. 2.

The calculated value that combines the three components of the adenyl system into a single formula and allows us to judge the direction of metabolic processes in the tissue / blood can be such a parameter as the energy charge of the adenyl system.

The energy charge of the adenyl system (in arbitrary units) was calculated by the formula: (ATP + 0.5 ADP) / (ATP + ADP + AMP) (Atkinson DE, Walton GM. Adenosine triphosphate conservation in metabolic regulation. Rat liver citrate cleavage enzyme // J. Biol. Chem. 1967, 242: 3239–3241).

For most healthy, normally functioning cells, this parameter is in the range of 0.80-0.95. A decrease in the parameter below 0.8 indicates a deterioration in the energy supply of body cells. When this value drops below 0.5, the cells die.

From the numerical values of this parameter presented in Table 3 in rat groups, the following conclusions can be drawn:

a) In group No. 2 (Control group of animals with bronchopulmonary inflammation), the value of this parameter is almost 40% less than the value of the same parameter in group No. 1 (Intact animals);

b) After the introduction of tetrapeptides, the values of these parameters (Groups No. 3 and No. 4) noticeably increase in comparison with the parameter value in Group No. 2;

c) In group No. 4 (Bronchopulmonary inflammation and administration of Ala-Glu-Asp-Leu-NH ₂ ), the value of this parameter is almost 19% higher than the value of the same parameter in group No. 3 (Bronchopulmonary inflammation and administration of Ala-Glu-Asp- Leu). Those. the introduction of the new tetrapeptide Ala-Glu-Asp-Leu-NH ₂ significantly more effectively normalizes the energy state of cells compared to the introduction of the peptide taken as a prototype.

Example 5. A model of chronic obstructive pulmonary disease (COPD) induced by cigarette smoke in rats.

Chronic obstructive pulmonary disease (COPD) in rats was modeled using the method (with some modifications) described in (Rodrigo de las and others. A new experimental model of cigarette smoke-induced emphysema in Wistar rats // J Bras Pneumol. 2014; 40 ( 1): 46-54).

A total of 48 male Wistar rats weighing 400-450 grams were taken for research. All animals were divided into eight equal in number of animals (in each group of 6 rats) groups:

1) Group No. 1 - intact animals;

2) Group No. 2 - animals with induced COPD and not receiving any treatment (negative control);

3) Group No. 3 - animals with induced COPD and additionally received an intragastric dose of 50 μg / kg of the new tetrapeptide;

4) Group No. 4 - animals with induced COPD and additionally received an intragastric dose of 500 μg / kg of a new tetrapeptide;

5) Group No. 5 - animals with induced COPD and additionally received an intragastric dose of 5000 μg / kg of the new tetrapeptide;

6) Group No. 6 - animals with induced COPD and additionally received an intravenous dose of 1 μg / kg of a new tetrapeptide;

7) Group No. 7 - animals with induced COPD and additionally received an intravenous dose of 10 μg / kg of the new tetrapeptide;

8) Group No. 8 - animals with induced COPD and additionally received an intravenous dose of 100 μg / kg of the new tetrapeptide.

Ala-Glu-Asp-Leu-NH ₂ tetrapeptide was used as a new test peptide in groups Nos. 3-8.

The induction of COPD was performed by fumigating experimental animals (except for a group of intact rats) with cigarette smoke using special equipment. A schematic diagram of this equipment is shown in FIG. 6.

The animal accommodation system consists of four metal booths (24 cm x 17 cm x 15 cm), closed with a grid on top. Each booth accommodates 3 rats. Metal cabins are symmetrically placed in an airtight, transparent, polymer box (70 cm x 70 cm x 20 cm). The study uses 2 experimental facilities. In one of these experimental installations animals are placed for fumigation with cigarette smoke. In another installation housed animals from the group of intact rats that were not exposed to cigarette smoke. Experimental facilities are located in various laboratory facilities. In laboratory premises maintained temperature within 20-25 ^° C ^and relative humidity 60-70%. The experimental setup consists of an external cigarette holder, which is connected by a flexible hose to a dynamic suction pump. The suction pump creates negative pressure, which causes clean air from the laboratory to pass through a lit cigarette to form cigarette smoke, which then flows through a hose into the polymer box. The suction pump is programmed to alternate periods of supply of cigarette smoke and periods of supply of clean air (to prevent asphyxiation in experimental animals) in the polymer box.

Five periods of exposure to cigarette smoke on experimental animals (except animals from the group of intact rats) were performed at approximately equal intervals of time per day. The duration of each period of exposure to cigarette smoke was 1 hour. During this hour, animals were exposed to smoke from 4 cigarettes. During this hour, fumigation with cigarette smoke was carried out for 15 minutes, then within 5 minutes clean air was supplied (to avoid hypoxia in animals). After that, the cycle of supplying cigarette smoke - supply of fresh air was repeated. The 15 minute period of animal fumigation with cigarette smoke, in turn, consisted of the following cycles: 15 seconds supply of cigarette smoke and 15 minutes - stop the supply of cigarette smoke. Thus, within 1 day, animals (except rats from the intact group) were exposed to cigarette smoke generated from the burning of 20 cigarettes.

The concentration of carbon monoxide (carbon monoxide) also contained in cigarette smoke in a polymer box was determined using a GasAlert Micro 5 portable gas detector (manufactured by Honeywell Analytics Distribution Inc). During the 1 hour period of exposure to cigarette smoke in animals, the concentration of this gas in the polymer box varied within 300-500 ppm. During the experiment, "Prima" cigarettes were used for fumigation with the following content of tar and nicotine in 1 cigarette - 16 mg and 1.2 mg, respectively. Taking into account the volume of the polymer box, the tar content in each cigarette, the number of cigarettes used during one day, the duration and number of periods the exposure of experimental animals to cigarette smoke, the average lung ventilation volume in rats, each animal (except for rats from the intact group) for one days received a dose of resin equal to 80 mg / kg

Table 4 shows the experimental design for male rats of the Wistar strain.

Table 4.

Design of an experiment on male Wistar rats in a model of chronic obstructive pulmonary disease (COPD) induced by cigarette smoke.

Group number Number 1 Number 2 Number 3 Number 4 Number 5 Number 6 Number 7 Number 8 The number of animals in the group, pieces 6 6 6 6 6 6 6 6 Route of administration No No Ext / F * Ext / F * Ext / F * Vn / v ** Vn / v ** Vn / v ** Duration of COPD induction, day No 24 24 24 24 24 24 24 Duration of drug administration from the beginning of COPD induction, day No No 10 10 10 10 10 10 Duration of drug administration, day No No fourteen fourteen fourteen fourteen fourteen fourteen The dose of the drug, mcg / kg No No fifty 500 5000 one 10 one hundred

Note: * intragastric route of administration of the drug; ** -Intravenous route of administration of the drug.

At the first stage of the study, animals from groups No. 2-8 were fumigated with cigarette smoke according to the method described above, for 10 days.

At the second stage of the study, animals of their groups No. 2-8 were additionally fumigated with cigarette smoke for another 14 days. However, animals from group No. 2 did not receive additional intravenous or intragastric new tetrapeptide. Animals from groups No. 3-5 were additionally intragastrically administered a new tetrapeptide in the doses indicated in Table 4. Animals from groups No. 6-8 additionally administered a new tetrapeptide at the doses indicated in Table 4.

In all groups nos. 3–8, sterile tetrapeptide solutions were used for administration. Sterile saline was used to prepare these solutions.

The volume of administration was 500 μl per rat with intragastric administration (groups No. 3-5) and 10 μl per rat with intravenous administration (groups 6-8).

At the first and second stage of the study, intact animals from group No. 1 were not fumigated with cigarette smoke, nor were they exposed to the introduction of a new tetrapeptide.

On the 28th day of the experiment, animals from all groups performed morphometric studies of pulmonary preparations (Example No. 7), as well as studies of the cellular composition of bronchoalveolar lavage (Example No. 8).

Example 6. Morphometric studies of preparations of rat lung tissue on a COPD model induced by cigarette smoke.

Morphometric studies of rat lung tissue preparations on a COPD model induced by cigarette smoke were performed for all eight experimental groups of animals.

For these studies, 3-4 animals were used in each group.

Before the study, the animals were subjected to general anesthesia using the combined preparation "Zoetil ^® 50" (manufactured by Virbac Sante Animale) (Dose 7 mg / kg).

The lungs were straightened by the introduction of a 10% solution of neutral formalin through the trachea. Conducted standard tissue fixation in formalin solution and dehydration in ethanol with increasing concentration (70–96%). After that, lung tissue samples were poured into paraffin. Histological sections 5–7 μm thick were made from paraffin blocks and stained with hematoxylin and eosin and according to Van Gieson.

Histological sections were photographed using a Nicon microscope and a Progres CF USB digital camera. Morphometric analysis was performed using the Video-Test-Morphology 5.2 hardware-software complex. Each drug was photographed with an increase of 4, 20 and 40 times. With a 4-fold increase, the bronchial wall was evaluated: the areas of the bronchus, cell infiltrate, bronchial lumen and the total area of other components of the bronchial wall (mucous and muscle membranes, connective tissue) were measured. The measurements were carried out using manual processing programs by highlighting the indicated structures and automatically determining their area (mm ² ) and perimeter (mm). The data obtained were used to calculate the areas of infiltrate.

The thickness of the muscle layer of the bronchial wall was evaluated at a 20-fold increase (measurement step 10 μm). Using manual processing programs, 10–20 consecutive measurements of each preparation were performed. The thickness of the interalveolar septa (μm) and the area of the alveoli (μm ² ) were determined at a 40-fold increase. In each preparation, using manual processing programs, 10–20 consecutive measurements of the thickness of the interalveolar septa were carried out. In the program of automatic measurements by binarization, the lumen of the alveoli was allocated and their area was determined.

Statistical analysis of the obtained experimental data was performed using the software "Statistics 6.0". The verification of the normality of the distribution of experimental data was performed using the Shapiro-Wilk test. Statistically significant differences between the mean values in each experimental sample were determined at a 0.05 significance level. The average values and standard errors in determining the average value for all experimental data according to the results of morphological studies for these groups of animals are shown below in table 5.

Table 5.

Results of morphometric studies of rat lung tissue preparations

Indicators Intact group Negative control Ext / w
50 mcg / kg Ext / w
500 mcg / kg Ext / w
5000 mcg / kg In / in
1 mcg / kg In / in
10 mcg / kg In / in
100 mcg / kg Area of infiltrate,% 11.5 ± 0.55 22.04 ± 0.62 (*) 19.8 ± 0.38 (*) 16.06 ± 0.45
(*), (**) 15.2 ± 0.60 (*), (**) 20.4 ± 0.65 (*) 18.64 ± 0.55 (*), (**) 14.6 ± 0.14, (**) The thickness of the muscle membrane of the wall of the bronchus, mm 8.9 ± 0.40 15.6 ± 0.33
(*) 14.2 ± 0.25
(*) 13.2 ± 0.30
(*), (**) 11.4 ± 0.45 (*), (**) 15.3 ± 0.55 (*) 12.4 ± 0.38 (*), (**) 9.8 ± 0.43
(**) The thickness of the interalveolar septum, mm 3.95 ± 0.25 4.98 ± 0.22
(*) 4.80 ± 0.24
(*) 4.65 ± 0.11
(*) 4.35 ± 0.18
(*), (**) 4.75 ± 0.18
(*) 4.58 ± 0.22
(*) 4.11 ± 0.08
(**) Alveolar lumen area, μm2 398.4 ± 32.0 489.5 ± 28.2 (*) 480.7 ± 38.0 (*) 470.5 ± 48.0 (**) 430.4 ± 45.0 (*), (**) 481.8 ± 45.0 (*) 467.7 ± 42.0
(*) 419.4 ± 38.0, (*), (**)

Note: (*) - the differences are statistically significant by the Student criterion (at p <0.05) compared with intact animals;

 (**) - the differences are statistically significant by Student's criterion (at p <0.05) compared with animals of negative control.

As can be seen from the data presented in table 5, in the lungs of animals from group No. 2 (negative control) compared with lung tissues from intact animals from group No. 1, there was a significant increase in the thickness of the muscular membrane of the bronchial wall, thickening of the interalveolar septa, and a significant increase in the percentage area occupied by cellular infiltrate. An increase in the lumen area of the alveoli was also noted.

With intragastric and intravenous administration of a new tetrapeptide in various doses to animals in groups No. 3-8, there is a tendency to improve all morphometric indicators compared with similar indicators in animals from group No. 2 (negative control). In some cases, the improvement of these indicators in animals in groups No. 3-8 are statistically significant compared with indicators in animals from group No. 2 (negative control).

 However, these indicators in most cases in animals in groups No. 3-8 did not reach intact values.

Therefore, the new tetrapeptide has a specific pharmacological effect, which manifests itself in a tendency to normalize and reduce the pathological processes that occur in the bronchopulmonary structures of animals with prolonged exposure to cigarette smoke.

Example 7. The study of the cellular composition of bronchoalveolar lavage of rats on a COPD model induced by cigarette smoke.

The cell composition of rat bronchoalveolar lavage (BAL) was determined on a COPD model induced by cigarette smoke for all eight experimental groups of animals.

For the collection of BAL, 3-4 animals were used in each group.

Before sampling bronchoalveolar lavage (BAL), the animals were subjected to general anesthesia using the combined preparation "Zoletil ^® 50" (manufactured by Virbac Sante Animale) (Dose 7 mg / kg).

In the animal, the intrathoracic trachea was exposed with a middle incision and an incision was made between the tracheal rings. Then a syringe was introduced with 5 ml of physiological saline. Saline solution was initially slowly, gradually squeezed out of the syringe, and then it was gradually taken into the syringe. After collecting BAL, the syringe was disconnected and the liquid thus obtained was transferred into 5 ml siliconized tubes. The BAL cell composition was examined using a BE-2800 Vet automatic veterinary hematology analyzer (manufactured by Mindray). The contents of the following cells were determined in the BAL of each animal: lymphocytes, macrophages, eosinophils, segmented neutrophils and segmented neutrophils.

Statistical analysis of the obtained experimental data was performed using the software "Statistics 6.0". The verification of the normality of the distribution of experimental data was performed using the Shapiro-Wilk test. Statistically significant differences between the mean values in each experimental sample were determined at a 0.05 significance level.

The average values and standard errors in determining the average value for all experimental data on the cellular composition of BAL for these groups of animals are shown below in table 6.

Table 6.

The cell composition of BAL experimental animals in absolute terms.

Indicators of the cellular composition of BAL, * 10 ⁹ / l Intact group Negative control Ext / w
50 mcg / kg Ext / w
500 mcg / kg Ext / w
5000 mcg / kg In / in
1 mcg / kg In / in
10 mcg / kg In / in
100 mcg / kg Lymphocytes 0.39 ± 0.11 1.34 ± 0.15 (*) 1.15 ± 0.18 (*) 1.06 ± 0.16 (*) 0.87 ± 0.15 (*) 0.95 ± 0.12 (*), (**) 0.64 ± 0.15 (*), (**) 0.58 ± 0.14, (**) Macrophages 0.034 ± 0.009 0.32 ± 0.01 (*) 0.21 ± 0.006 (*), (**) 0.16 ± 0.008 (*), (**) 0.12 ± 0.01 (*), (**) 0.25 ± 0.009 (*), (**) 0.21 ± 0.01 (*), (**) 0.09 ± 0.007 (*), (**) Eosinophils 0.014 ± 0.005 0.033 ± 0.007 0.025 ± 0.006 0.022 ± 0.007 (*) 0.019 ± 0.01 0.024 ± 0.005 0.022 ± 0.006 0.018 ± 0.007 Segmented neutrophils 0.28 ± 0.07 1.31 ± 0.18 (*) 0.65 ± 0.08 (*), (**) 0.48 ± 0.09, (**) 0.39 ± 0.05, (**) 0.84 ± 0.11 (*), (**) 0.55 ± 0.06
(**) 0.38 ± 0.08, (**) Stab neutrophils 0.008 ± 0.004 0.042 ± 0.008 (*) 0.030 ± 0.009 (*) 0.028 ± 0.012 (*) 0.016 ± 0.007, (**) 0.025 ± 0.01 (*), (**) 0.015 ± 0.006, (**) 0.012 ± 0.005 (**) Common cytosis 0.73 ± 0.19 3.16 ± 0.36 (*) 2.07 ± 0.28 (*), (**) 1.75 ± 0.27 (*), (**) 1.42 ± 0.23 (*), (**) 2.10 ± 0.25 (*), (**) 1.44 ± 0.23 (*), (**) 1.08 ± 0.25 (*), (**)

Note: (*) - the differences are statistically significant by the Student criterion (at p <0.05) compared with intact animals;

 (**) - the differences are statistically significant by Student's criterion (at p <0.05) compared with animals of negative control.

As can be seen from the data presented in table 6, in the animals in the negative control group, compared with animals from the intact group, the cellular composition of BAL is noticeably changed - there is a statistically significant increase in the content of: lymphocytes, macrophages and both types of neutrophils. In addition, there is a statistically significant increase in total cytosis of BAL. Such a cellular composition of BAL indicates the presence of bronchial inflammation in animals from the negative control group.

A similar picture, indicating the presence of inflammatory processes, is observed in other experimental groups of animals with COPD, but who received an additional tetrapeptide. However, the severity of inflammatory processes in these groups of animals is noticeably weaker. This is especially evident when comparing the relative values of the BAL cell composition for various animal groups with respect to the similar values of the BAL cell composition in the intact group of animals shown in Table 7.

Table 7.

The cellular composition of BAL experimental animals in relative values relative to the corresponding values in the intact group

Indicators of the cellular composition of BAL, in% Intact group Negative control Ext / w
50 mcg / kg Ext / w
500 mcg / kg Ext / w
5000 mcg / kg In / in
1 mcg / kg In / in
10 mcg / kg In / in
100 mcg / kg Lymphocytes one hundred %
(0.39 ± 0.11) 243 195 172 123 143 64 49 Macrophages one hundred %
(0,034 ± 0,009) 841 517 370 252 635 517 76 Eosinophils one hundred %
(0.014 ± 0.005) 136 78 57 36 71 57 28 Segmented neutrophils one hundred %
(0.28 ± 0.07) 368 132 71 39 200 96 36 Stab neutrophils one hundred %
(0.008 ± 0.004) 425 275 250 160 213 88 fifty Common cytosis one hundred %
(0.73 ± 0.19) 333 184 140 94 188 97 45

Table 7 shows the relative values of the BAL cell composition in various groups of animals with respect to the similar values of the BAL cell composition in the intact group of animals. The cellular composition of BAL in the intact group of animals was taken as 100%.

Therefore, the new tetrapeptide has a specific pharmacological effect, which manifests itself as a decrease in the inflammatory process in the mucous membranes of the bronchi of animals, as well as a tendency to normalize the cellular composition of bronchoalveolar lavage of these animals.

In addition, as can be seen from the experimental data presented in tables 6 and 7, with an increase in the dose of the tetrapeptide administered (both with intragastric and intravenous methods of administration), there is a fairly pronounced tendency to a decrease in inflammatory processes in the bronchial mucous membranes in groups of animals with COPD who received additional new tetrapeptide.

Example 8. The effect of the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ on the growth of explants of organotypic rat lung culture.

To determine the effect of the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ on the growth of explants of organotypic culture of rat lungs, the technique described in (Al-Shakri S.Kh., Chalisova N.I., Zakutsky A.N., Borovets S .U. Effect of amino acids and their metabolites on the development of organotypic testis culture in rats // Nephrology. 2007; 11 (2): 86-92).

Fresh tissues of rats of Wistar male rats at the age of 3-4 months were divided into small fragments of about 1 mm ³ . Thus obtained pieces of lungs of rats (explants) were used to prepare samples of the experimental group and samples of the control group. To prepare samples of the experimental and control groups, pieces of rat lungs were placed in a collagen-coated Petri dish (approximately 10-15 explants) in 1 Petri dish, and then 15 ml of culture medium were added for cultivation.

The composition of the nutrient medium used (based on 100 ml): Hanks solution - 41 ml; Medium Needle -30 ml, fetal calf serum - 25 ml, aqueous solution of 20% glucose -1 ml, insulin -2.5 ml (100 PIECES) and gentamicin 0.5 ml (20 mg). A culture medium was used for research, the pH of which varied in the range 7.2–7.4.

In the experimental group, the test solution of the new tetrapeptide was added to Petri dishes to the mixture of the explant and culture medium in various amounts, so that the concentration of this compound in the culture medium varied in the range of 0.01-10 ng / ml of culture medium. The volume of the added tetrapeptide solution in Petri dishes was 15 μl.

In the control group with explant culture medium, instead of the tetrapeptide solution, 15 μl of physiological saline was added to Petri dishes. After that, the contents of Petri dishes from the experimental group (with explants, nutrient medium and tetrapeptide solution) and the control group (with explants, nutrient medium and physiological saline) were cultured in a laboratory CO ₂ incubator with strict observance of temperature (36.5 ± 0, 25 ^O C) mode in a gaseous environment with a content of 5% carbon dioxide at a relative humidity of 95 ± 5%.

Observation of changes in cell structures in Petri dishes was carried out using an OLYMPUS CKX41 microscope with a VIDI-CAM digital imaging module installed. Processing of the obtained images was carried out using the IMAG PRO INSIGHT program. After 3 days of cultivation, two zones were observed in the explants of the control and experimental groups: central and peripheral. The central zone (with a higher optical density) represents rat lung cells originally placed on a collagen coating. The peripheral zone (growth zone) is formed by cells that migrate from the source tissues of the explants, and then proliferate on the collagen coating.

 To quantify the growth of explant cells, the parameter "Area Index (PI)" was determined, expressed in arbitrary units and calculated by the following formula:

PI = (The area of the entire explant) / (The area of the central zone of the explant).

To process the obtained IP measurement results, mathematical swastika methods were used. The area indices for the explants from the control (IPcon) and experimental (IP experience) groups were calculated. As an indicator, reflecting the solution of the new tetrapeptide on the growth of explants of organotypic culture of the lungs of rats, the percentage of experience / IPcon was used.

Table 8 shows the IPper / IPcon values with a change in the concentration of the new tetrapeptide 0.7-10 μg / ml of culture medium.

Table 8.

The effect of various concentrations of the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ in the culture medium on the growth of explants of organotypic culture of rat lungs.

Name of indicator The concentration of the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ in the culture medium, ng / ml 0.01 0.5 2,5 5,0 10.0 Experience / IPcon,% 3 19.0 * 37.0 * 45.0 * 55.0 *

Note: * statistically significant differences compared with the control at p <0.05.

The experimental results are shown in table 8, from which it follows that the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ significantly stimulates the growth of explants of organotypic culture of rat lungs in a wide range of concentrations from 0.5 to 10.0 ng / ml.

Thus, the use of the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ with a direct effect on the organotypic tissue culture of lung rats promotes accelerated renewal of the cell population of these tissues.

Example 9. The effect of tetrapeptides Ala-Glu-Asp-Leu-NH ₂ and Ala-Glu-Asp-Leu on the formation of an immune response to a thymus-dependent antigen.

The study of tetrapeptides Ala-Glu-Asp-Leu-NH ₂ and Ala-Glu-Asp-Leu on the formation of an immune response to a thymus-dependent antigen was performed on male CBA mice and weighing 20-30 g.

Studies were performed on 28 animals. All animals were divided into the following 7 equal groups of animals (4 animals in each group):

Group No. 1. (Control group). Immunized animals with additional saline.

Group No. 2. Animals immunized with additional intramuscular injection of the tetrapeptide Ala-Glu-Asp-Leu at a dose of 0.1 μg / kg;

Group No. 3. Animals immunized with additional intramuscular injection of the tetrapeptide Ala-Glu-Asp-Leu at a dose of 10 μg / kg;

Group No. 4. Animals immunized with additional intramuscular injection of the tetrapeptide Ala-Glu-Asp-Leu at a dose of 1000 μg / kg;

Group No. 5. Animals immunized with additional intramuscular injection of the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ at a dose of 0.1 μg / kg;

Group No. 6. Animals immunized with additional intramuscular injection of the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ at a dose of 10 μg / kg;

Group No. 7. Animals immunized with additional intramuscular injection of the tetrapeptide Ala-Glu-Asp-Leu-NH ₂ at a dose of 1000 μg / kg

The animals from the experimental groups No. 2-7 6 days before immunization were injected intramuscularly daily with solutions of the studied peptides in an amount of 0.1 ml per animal. For administration, sterile tetrapeptide solutions were used. Sterile saline was used to prepare these solutions.

Animals from the control group during these 6 days (group No. 1) were injected with a sterile isotonic sodium chloride solution.

The animal was immunized by a single intravenous administration of 0.05 ml of a suspension of sheep erythrocytes in sterile physiological saline (dose of 5 · 10 ⁷ sheep erythrocytes / mouse).

On the 5th day after immunization, blood was obtained from animals by decapitation to obtain serum and determine the values of hemagglutinin titers (GMA). Then the animals were euthanized using cervical dislocation. After that, the spleens were removed and a cell suspension of the organ was prepared for the quantitative determination of antibody-forming cells (AOK) using the method of Erne and Nordin.

The obtained experimental data (numerical values of AOK and GMA) were subjected to statistical processing using the software "Statistics 6.0". The verification of the normality of the distribution of experimental data was performed using the Shapiro-Wilk test. Statistically significant differences between the mean values in each experimental sample were determined at a 0.05 significance level. The average values and standard errors in determining the average value for all experimental data on titers of GMA and AOK for all groups of animals are shown below in table 9.

Table 9.

The effect of tetrapeptides Ala-Glu-Asp-Leu-NH ₂ and Ala-Glu-Asp-Leu on the immune response in CBA mice

Group number Used tetrapeptide Dose, mcg / kg Immune Response GMA titers, log ₂ The number of AOK per 10 ⁶ cells of the spleen one Control group - / - 7.9 ± 0.5 135.1 ± 8.2 2 Ala-Glu-Asp-Leu 0.1 8.1 ± 0.3 138.1 ± 20.2 3 Ala-Glu-Asp-Leu 10.0 8.6 ± 0.4 142.6 ± 18.2 four Ala-Glu-Asp-Leu 1000,0 8.7 ± 0.6 143.2 ± 14.5 5 Ala-Glu-Asp-Leu-NH ₂ 0.1 8.9 ± 0.3 * 206.4 ± 18.5 * 6 Ala-Glu-Asp-Leu-NH ₂ 10.0 12.4 ± 0.5 * 458.4 ± 41.3 * 7 Ala-Glu-Asp-Leu-NH ₂ 1000,0 14.8 ± 0.6 * 472.5 ± 31.2 *

Note: * significant differences with control were noted (p <0.05).

Abbreviations used: GMA - hemagglutinins, AOK - antibody-forming cells.

As follows from the data presented in table 9, the use of the new tetrapeptide Ala-Glu-Asp-Leu-NH ₂ leads to a significant increase in the number of antibody-forming splenocytes (AOKs) and anti-erythrocyte antibodies (GMA). Significant differences with the control in the group where the new Ala-Glu-Asp-Leu-NH ₂ tetrapeptide was used were already noted when the substance was administered at a dose of 0.1 μg / kg. In animals that were injected with the Ala-Glu-Asp-Leu tetrapeptide, the effects of increasing the amounts of AOA and GMA are very weak. These results indicate that, in contrast to the prototype (Ala-Glu-Asp-Leu tetrapeptide), the new tetrapeptide (Ala-Glu-Asp-Leu-NH ₂ ) has a pronounced immunomodulating activity.

Thus, the claimed peptide compound has a comprehensive restorative effect on the function of the respiratory system, including immunomodulation.

Claims (3)

1. The tetrapeptide alanyl-glutamyl-aspartyl-leucine amide of the general formula Ala-Glu-Asp-Leu-NH ₂ . 2. The use of the tetrapeptide alanyl-glutamyl-aspartyl-leucine amide of the general formula Ala-Glu-Asp-Leu-NH ₂ to restore respiratory function and immunocorrection. 3. Pharmacological agent of a peptide nature, restoring the function of the respiratory system and having an immunomodulating effect, containing an active peptide agent and a pharmaceutically acceptable carrier, characterized in that as an active peptide agent contains an effective amount of a tetrapeptide Ala-Glu-Asp-Leu-NH ₂ in a dose 0.01-1000 mcg / kg body weight.

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