RU2790871C1 - USE OF H2N-D-Ala-L-Glu-L-Trp-COOH PEPTIDE FOR HEPATOPROTECTIVE EFFECTS. - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: invention relates to the use of a peptide of the formula H2N-D-Ala-L-Glu-L-Trp-COOH for hepatoprotective effects and can be used in medicine.

EFFECT: using peptide H2N-D-Ala-L-Glu-L-Trp-COOH in case of liver damage to provide antioxidant and reparative effect on hepatocytes, activate the reparative regeneration of hepatocytes, increase the number of their mitoses in the liver, and reduce the level of lipid peroxidation product — malondialdehyde in liver cells.

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Description

The invention relates to medicine (pharmacology, hepatology, pathophysiology), veterinary medicine and can be used to stimulate the antioxidant defense and reparative activity of hepatocytes, to achieve a hepatoprotective effect when using the peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH under conditions liver damage.

Known dipeptide H ₂ NL-Glu-L-Trp-COOH, which is used in medicine as an immunomodulating agent. It is called thymogen and has a number of other biological effects (Register of Medicines of Russia (RLS) Encyclopedia of Medicines, 2004, 11th edition, p. 849-850).

Peptides may have a short duration of their effects due to inactivation by proteases, which are elevated during inflammation (Robson, M.C. Wound infection: a failure of wound healing caused by an imbalance of bacteria / M.C. Robson // Surg. Clin. North. Am. - 1997. - Vol. 77. - P. 637-650.), (Saidakhmedova, Z.T. Immunomodulating activity of thymogen in acute pancreatitis / Z.T. Saidakhmedova // Vopr. Pitan. - 2000. - T.69, N 6. - P 35-36.) The introduction of D-amino acids into the molecule is considered one of the ways to increase the resistance of peptides to the action of proteases (Greven H.M., De Wied D. Influence of peptides structurally related to ACTH and MSH on active avoidance in rats // Frontiers of Hormone research 1977, v.4, p.140-152)

Structural similarity of peptides is not a basis for the similarity of their biological effects. It is known that the replacement of an L-amino acid with a D-amino acid often leads to a change in the direction of the effects of the peptide in the opposite direction or their complete leveling (Deigin, Vladislav. EW-peptide family. One family, two drugs / Vladislav Deigin // Journal of peptide science - 2008. - V. 14. - Issue 8. - P.163.). These patterns are especially characteristic of short peptides (Ashmarin I.P., Antonova JI.B., Titov S.A., Maksimova JI.A., Kamensky A.A. Possible mechanisms of the multidirectional action of ACTH _4-10 and its analogue containing D-isomer of phenylalanine, on behavior // Journal of VND, 1980, vol. Therefore, with such modifications of molecules as the change of L-amino acids to their D-isomers, it is impossible to predict in advance how their effects will change in comparison with the original molecule. And in this case, we can assume the manifestation of new biological effects.

Thus, when the amino acid D-Ala (D-alanine) is introduced into the thymogen molecule from the N-terminus, the immunostimulatory, antioxidant and reparative activities are enhanced. Peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH in conditions of skin wounds had more pronounced immunostimulatory, antioxidant and wound healing effects compared to thymogen (H ₂ NL-Glu-L-Trp-COOH) (Wound healing agent, Koroshvili V.T., Smakhtin M.Yu., Bobyntsev I.I., Ardemasova Z.A., Kalikhevich V.N., Litvinova E.S. Eurasian Patent No. EA201400777A1 20160129, issued January 30 2017).

Data on the effect of the peptide of the general formula H ₂ ND-Ala-L-Glu-L-Trp-COOH on the state of the liver and its antioxidant activity in pathologies of this organ are not available in the literature. This study was carried out for the first time.

The technical result of the invention: the use of peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH for a new purpose to increase antioxidant and reparative activities and to achieve a hepatoprotective effect in liver damage.

The technical result of the invention is achieved by using the H ₂ ND-Ala-L-Glu-L-Trp-COOH peptide to increase antioxidant and reparative activities and to achieve a hepatoprotective effect in liver lesions.

The peptide can be synthesized in any peptide synthesis laboratory from single amino acids. We used a preparation synthesized at the Research Institute of Chemistry of St. Petersburg State University.

Peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH was dissolved in isotonic sodium chloride solution and administered to experimental animals (rats) once a day parenterally (intraperitoneally) in a single dose of 1.2 μg/kg for 5 days. Carbon tetrachloride and hydrazine were used as hepatotropic poisons. The peptide was administered to animals in a volume of 0.1 ml once a day with an interval of 24 hours between injections. The antioxidant activity of the peptide was determined by the level of malondialdehyde (MDA) in blood plasma and liver homogenate (Ragino, Yu. I. Determination of resistance to oxidation of heparin-precipitated β-lipoproteins in blood serum in patients with coronary heart disease / Yu. I. Ragino, M. I. Dushkin // Clinical laboratory diagnostics. - 1998. - No. 11. - P. 3-5). A portion of the liver was placed in a tenfold volume of ice-cold saline and homogenized. The obtained homogenate was centrifuged for 10 minutes at a speed of 3000 rpm and the resulting supernatant was used for research. MDA is the end product of lipid peroxidation (Mihara, M. Thiobarbituric acid value on fuch homogenate of rat as a parameter of lipid peroxidation in aging, CCl ₄ intoxication and vitamin E definienes / M. Mihara, M. Uchiama // Biochem. Med. - 1980. - Vol. 23, No. 3. - P. 302-311). The mitotic index (MI) of hepatocytes was calculated, characterizing the activity of their reparative regeneration. Statistical processing of the results was carried out by the divergence of the boundaries of confidence intervals and using the Mann-Whitney test.

Changes in the level of MDA in the liver and reparative regeneration of hepatocytes are also used in determining hepatotoxicity or hepatoprotective effects of pharmacological drugs and serve as indicators of the state of the liver (Podymova, S.D. Liver diseases / S.D. Podymova. - M., 1993. - 544 p. Guidelines for the experimental (preclinical) study of new pharmacological substances. - M .: Ministry of Health of the Russian Federation, CJSC IIA "Remedium", 2000.)

An example of a specific implementation.

Example 1. Effects of peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH in carbon tetrachloride poisoning.

Wistar rats were modeled with carbon tetrachloride liver damage by intragastric administration of its 50% solution in vegetable oil at 3 ml/kg of body weight for 5 consecutive days. A five-day administration of carbon tetrachloride is accompanied by medium droplet fatty degeneration of hepatocytes (Pharmacological correction of immunometabolic disorders with the peptide GLY-HIS-LYS in conditions of liver damage with carbon tetrachloride / M.Yu. Smakhtin, A.I. Konoplya, L.A. Severyanova, I.A. Shveinov // Pathological physiology and experimental therapy. - 2003. - No. 2. - P. 19-21.).

A portion of the peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH was dissolved in a sterile isotonic sodium chloride solution and administered to animals in a volume of 0.1 ml intraperitoneally at a single dose of 1.2 μg/kg body weight five times (once a day ) simultaneously with the introduction of carbon tetrachloride. Animals of the control group received equivalent volumes of isotonic solution (NaCl). The next day after the last injection, the animals were taken out of the experiment by exsanguination under chloral hydrate anesthesia.

The antioxidant activity of the peptide was determined by the level of malondialdehyde (MDA) in blood plasma and liver homogenate (Ragino, Yu. I. Determination of resistance to oxidation of heparin-precipitated β-lipoproteins in blood serum in patients with coronary heart disease / Yu. I. Ragino, M. I. Dushkin // Clinical laboratory diagnostics. - 1998. - No. 11. - P. 3-5). MDA is the end product of lipid peroxidation (Mihara, M. Thiobarbituric acid value on fuch homogenate of rat as a parameter of lipid peroxidation in aging, CCl ₄ intoxication and vitamin E definienes / M. Mihara, M. Uchiama // Biochem. Med. - 1980. - Vol. 23, No. 3. - P. 302-311). Also, liver pieces were fixed in 10% neutral formalin in 0.1 M phosphate buffer pH=7.2 and embedded in paraffin according to the standard method. Paraffin liver sections were stained with hematoxylin and eosin. The mitotic index (MI) of hepatocytes was calculated, characterizing the activity of their reparative regeneration (Sarkisov, D.S. Microscopic technique / D.S. Sarkisov. - M.: Medicine, 1996. - 543 p.).

The revealed effects are presented in Table 1.

Table 1 Influence of peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH on MDA concentration and mitotic index (M±m, n=8) of hepatocytes under conditions of carbon tetrachloride poisoning animal groups Antioxidant activity Reparative activity MDA, µmol/l in blood plasma MDA, µmol/l in
liver homogenate Mitotic index of hepatocytes, ‰ 1.Control (NaCl) 6.1±0.6 6.9±0.7 2.5±0.1 2. _H2ND -Ala-L-Glu-L-Trp-COOH 2.3±0.3* 4.8±0.5* 12.3±0.3*

Note: * - shows significant differences, p<0.05; n is the number of animals in the group.

Peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH reduces the level of malondialdehyde (MDA) in blood plasma and in the liver (homogenate), which indicates its antioxidant activity (table 1). Also, the peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH activates the reparative regeneration of hepatocytes, increasing the number of their mitoses in the liver and thereby activating the restoration of the organ affected by carbon tetrachloride. A decrease in the level of MDA in the liver and an increase in the mitotic index of hepatocytes indicate the manifestation of a hepatoprotective effect.

Example 2 Effects of the H ₂ ND-Ala-L-Glu-L-Trp-COOH peptide on hydrazine poisoning.

As another model of acute toxic hepatopathy, liver damage was also used by a single intraperitoneal injection of hydrochloric acid hydrazine (50 mg / kg) (Ivanova, A.P. Formation of the immune response and its correction in toxic liver damage of various etiologies: Dis. ... candidate of medical sciences / A.P. Ivanova. - Kursk, 1995. - 140 p.). A portion of the peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH was dissolved in a sterile isotonic sodium chloride solution and administered to animals in a volume of 0.1 ml intraperitoneally at a single dose of 1.2 μg/kg body weight five times (once a day ) simultaneously and after the introduction of hydrazine. Animals of the control group received equivalent volumes of isotonic solution (NaCl). The next day after the last injection, the animals were taken out of the experiment by exsanguination under chloral hydrate anesthesia. The same indicators were determined. The revealed effects are presented in Table 2.

Peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH reduces the level of malondialdehyde (MDA) in blood plasma and in the liver (homogenate), which indicates its antioxidant activity (table 2). Also, the peptide H ₂ ND-Ala-L-Glu-L-Trp-COOH activates the reparative regeneration of hepatocytes, increasing the number of their mitoses in the liver and thereby activating the restoration of the organ affected by hydrazine. A decrease in the level of MDA in the liver and an increase in the mitotic index of hepatocytes indicate the manifestation of a hepatoprotective effect.

table 2 Effect of H ₂ ND-Ala-L-Glu-L-Trp-COOH peptide on MDA concentration and mitotic index (M±m, n=8) of hepatocytes under conditions of hydrazine poisoning animal groups Antioxidant activity Reparative activity MDA, µmol/l in blood plasma MDA, µmol/l in
liver homogenate Mitotic index of hepatocytes, ‰ 1.Control (NaCl) 5.1±0.5 6.3±0.6 2.1±0.1 2. _H2ND -Ala-L-Glu-L-Trp-COOH 3.6±0.3* 3.4±0.4* 22.6±0.6*

Note: * - shows significant differences, p<0.05; n is the number of animals in the group.

Thus, the peptide having the formula H ₂ ND-Ala-L-Glu-L-Trp-COOH has antioxidant effects, stimulates the processes of reparative regeneration of hepatocytes, increasing the number of their mitoses, and therefore activates the process of liver repair, thereby exhibiting hepatoprotective activity .

Claims (1)

The use of a peptide of the formula H ₂ ND-Ala-L-Glu-L-Trp-COOH, which has antioxidant and reparative effects for hepatoprotective effects in liver damage.